Electron microscopic localization and experimental modification of NADPH-diaphorase activity in crustacean sensory axons

The origin and ultrastructural localization of NADPH-diaphorase (NADPH-d) in the olfactory afferent pathway of the crayfishPacifastacus leniusculus was investigated by means of histochemical techniques. Sensory axons in the antennular nerve and the olfactory lobe glomeruli of normal animals expressed NADPH-d staining properties. The NADPH-d staining of each glomerulus was regionalized showing pronounced staining in the apical cap-region. Following ablation of the chemosensory input for 30 days, the staining properties of the antennular nerve and the glomeruli were reduced. At the electron microscopic level, the NADPH-d precipitate was found to be distributed on various membranes in neuronal profiles and glial cells. Stained neuronal profiles were frequently observed in the glomeruli, whereas the number of positive glial cells was low. Almost all NADPH-d positive profiles in the neuropil had an intraglomerular localization. The present findings suggest that NADPH-d in the crayfish olfactory lobe neuropil is localized to terminals of olfactory sensory axons.     

Localization of nerve growth factor-like immunoreactivity in rat nervous tissue

The use of immunofluorescence with affinity-purified antibodies enabled cytological localization of nerve growth factor-like material in the rat. Immunoreactivity was observed along various nerve tracts of the foetal rat brain and spinal cord at day 15 of gestation. Longitudinal pathways in ventral and dorsal spinal cord, ventral lower brain stem, posterior commissure, retroflex fascicle and in the olfactory bulb were all positive. A weaker and more widely spread immunostaining was visible in many areas in the central nervous system. Cranial nerves were strongly immunoreactive. Neuronal perikarya in the retina and the olfactory mucosa as well as filae olfactoriae and the olfactory nerve all the way to the olfactory bulb were also positive. In sensory ganglia and peripheral nerves most immunoreactivity was confined to supporting tissues, probably including Schwann cells. In irides, the pattern of immunoreactivity was similar to that of the sensory and autonomic innervation. More intensively fluorescent material was found in regrowing nerve fibres in iris transplants. Our histochemical results suggest that nerve growth factor and/or a related protein is present in large amounts along nerve pathways in supportive tissues of the peripheral nervous system as well as in the central nervous system during early development.     

Expression of cell adhesion molecules in the olfactory system of the adult mouse: presence of the embryonic form of N-CAM

The expression of the neural cell adhesion molecules N-CAM and L1 was investigated in the olfactory system of the mouse using immunocytochemical and immunochemical techniques. In the olfactory epithelium, globose basal cells and olfactory neurons were stained by the polyclonal N-CAM antibody reacting with all three components of N-CAM (N-CAM total) in their adult and embryonic states. Dark basal cells and supporting cells were not found positive for N-CAM total. The embryonic form of N-CAM (E-N-CAM) was only observed on the majority of globose basal cells, the precursor cells of olfactory neurons, and some neuronal elements, probably immature neurons, since they were localized adjacent to the basal cell layer. Differentiated neurons in the olfactory epithelium did not express E-N-CAM. In contrast to N-CAM total, the 180-kDa component of N-CAM (N-CAM180) and E-N-CAM, L1 was not detectable on cell bodies in the olfactory epithelium. L1 and N-CAM180 were strongly expressed on axons leaving the olfactory epithelium. Olfactory axons were also labeled by antibodies to N-CAM180 and L1 in the lamina propria and the nerve fiber and glomerular layers of the olfactory bulb, but only some axons showed a positive immunoreaction for E-N-CAM. Ensheathing cells in the olfactory nerve were observed to bear some labeling for N-CAM total, L1, and N-CAM180, but not E-N-CAM. In the olfactory bulb, L1 was not present on glial cells. In contrast, N-CAM180 was detectable on some glia and N-CAM total on virtually all glia. Glia in the nerve fiber layer were labeled by E-N-CAM antibody only at the external glial limiting membrane. In the glomerular layer, E-N-CAM expression was particularly pronounced at contacts between olfactory axons and target cells. The presence of E-N-CAM in the adult olfactory epithelium and bulb was confirmed by Western blot analysis. The continued presence of E-N-CAM in adulthood on neuronal precursor cells, a subpopulation of olfactory axons, glial cells at the glia limitans, and contacts between olfactory axons and their target cells indicates the retention of embryonic features in the mammalian olfactory system, which may underlie its remarkable regenerative capacity.     

The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform

Although N-CAM has previously been implicated in the growth and fasciculation of axons, the development of axon tracts in transgenic mice with a targeted deletion of the 180-kD isoform of the neural cell adhesion molecule (N-CAM-180) appears grossly normal in comparison to wild-type mice. We examined the organization of the olfactory nerve projection from the olfactory neuroepithelium to glomeruli in the olfactory bulb of postnatal N-CAM-180 null mutant mice. Immunostaining for olfactory marker protein revealed the normal presence of fully mature primary olfactory neurons within the olfactory neuroepithelium of mutant mice. The axons of these neurons form an olfactory nerve, enter the nerve fiber layer of the olfactory bulb, and terminate in olfactory glomeruli as in wild-type control animals. The olfactory bulb is smaller and the nerve fiber layer is relatively thicker in mutants than in wild-type mice. Previous studies have revealed that the plant lectin Dolichos biflorus agglutinin (DBA) clearly stains the perikarya and axons of a subpopulation of primary olfactory neurons. Thus, DBA staining enabled the morphology of the olfactory nerve pathway to be examined at higher resolution in both control and mutant animals. Despite a normal spatial pattern of DBA-stained neurons within the nasal cavity, there was a distorted axonal projection of these neurons onto the surface of the olfactory bulb in N-CAM-180 null mutants. In particular, DBA-stained axons formed fewer and smaller glomeruli in the olfactory bulbs of mutants in comparison to wild-type mice. Many primary olfactory axons failed to exit the nerve fiber layer and contribute to glomerular formation. These results indicate that N-CAM-180 plays an important role in the growth and fasciculation of primary olfactory axons and is essential for normal development of olfactory glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 643–658, 1997     

A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry

The localization of acetylcholinesterase (AChE) as revealed either by enzyme-histochemical or by immunohistochemical methods was compared in distinct regions of the rat brain. In general, the localization of AChE observed was nearly the same, whether revealed by histochemical demonstration of its catalytic activity or by immunohistochemical detection of the enzyme molecule itself, in all regions investigated. Penetration problems of the antibodies, however, arose on strong myelin sheaths of the facial nerve, for instance, where no immunohistochemical staining was found though there was a relatively strong histochemical reaction. These problems could be partly solved by increasing the normal concentration of Triton X-100 added to the immunohistochemical solutions (0.1%) to 2.5%. Furthermore, it seems that sites containing low amounts of AChE could be better detected by the enzyme-histochemical method, whereas the depiction of structures (particularly of nerve fibres) was somewhat sharper with the immunohistochemical method.     

Expression of extracellular matrix molecules in the embryonic rat olfactory pathway

Primary olfactory neurons arise from placodal neuroepithelium that is separate from the neuroepithelial plate that forms the neural tube and crest. The axons of these neurons course along a stereotypical pathway and invade the rostral telencephalic vesicle where they induce the formation of the olfactory bulb. In the present study we examined the expression of several extracellular matrix constituents during formation of the olfactory nerve pathway in order to identify putative developmentally significant molecules. Double-label immunofluorescence was used to simultaneously map the trajectory of growing primary olfactory axons by expression of growth associated protein 43 (GAP-43) and the distribution of either laminin, heparan sulfate proteoglycans (HSPG), or chondroitin sulfate proteoglycans (CSPG). At embryonic day 12.5 (E12.5) primary olfactory axons have exited the olfactory neuroepithelium of the nasal pit and formed a rudimentary olfactory nerve. These axons together with migrating neural cells form a large mass outside the rostral surface of the telencephalon. This nerve pathway is clearly defined by a punctate distribution of laminin and HSPG. CSPG is selectively present in the mesenchyme between the olfactory nerve pathway and the nasal pit and in the marginal zone of the telencephalon. At E14.5 primary olfactory axons pierce the telencephalon through gaps that have emerged in the basement membrane. At this age both laminin and HSPG are colocalized with the primary olfactory axons that have entered the marginal zone of the telencephalon. CSPG expression becomes downregulated in this same region while it remains highly expressed in the marginal zone adjacent to the presumptive olfactory bulb. By E16.5 most of the basement membrane separating the olfactory nerve from the telencephalon has degraded, and there is direct continuity between the olfactory nerve pathway and the central nervous system. This strict spatiotemporal regulation of extracellular matrix constituents in the olfactory nerve pathway supports an important role of these molecules in axon guidance. We propose that laminin and HSPG are expressed by migrating olfactory Schwann cells in the developing olfactory nerve pathway and that these molecules provide a conducive substrate for axon growth between the olfactory neuroepithelium and the brain. CSPG in the surrounding mesenchyme may act to restrict axon growth to within this pathway. The regional degradation of the basement membrane of the telencephalon and the downregulation of CSPG within the marginal zone probably facilitates the passage of primary olfactory axons into the brain to form the presumptive nerve fiber layer of the olfactory bulb. © 1996 John Wiley & Sons, Inc.     

Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb

In early rat embryos when axons from sensory neurons first contact the olfactory bulb primordium, lactosamine-containing glycans (LCG) are detected on neurons that are broadly distributed within the olfactory epithelium, but that project axons to a very restricted region of the ventromedial olfactory bulb. LCG(+) axons extend through channels defined by the coexpression of galectin-1 and beta2-laminin. These two extracellular matrix molecules are differentially expressed, along with semaphorin 3A, by subsets of ensheathing cells in the ventral nerve layer of the olfactory bulb. The overlapping expression of these molecules creates an axon-sorting domain that is capable of promoting and repelling subsets of olfactory axons. Specifically, LCG(+) axons preferentially grow into the region of the nerve layer that expresses high amounts of galectin-1, beta2-laminin, and semaphorin 3A, whereas neuropilin-1(+) axons grow in a complementary pattern, avoiding the ventral nerve layer and projecting medially and laterally. These studies suggest that initial patterning of olfactory epithelium to olfactory bulb connections is, in part, dependent on extracellular components of the embryonic nerve layer that mediate convergence and divergence of specific axon subsets.     

NADPH-Diaphorase in the Olfactory System of the Carp Cyprinus carpio

Ultrastructural distribution of NADPH-diaphorase (NADPH-d) in olfactory epithelium and bulb of the carp Cyprinus carpio L. was studied using light and electron microscopy. The diaphorase staining was revealed in the supranuclear area of the sensory and indifferent epithelium, in the olfactory nerve, as well as in the outer layers of the olfactory bulb—in fibers and glomeruli. NADPH-d-positive neurons were found in the interglomerular neuropil. Electron microscopy showed that NADPH-d in the olfactory lining epithelium was related only to receptor cells and ciliary supporting cells and was present in submembranous structures. Besides, in both parts of the olfactory system the main, cytosolic part of the enzyme is bound to cytoskeleton and is also present in membranes of endoplasmic reticulum and in mitochondria. In general, the NADPH-d of the carp olfactory system is characterized by predominantly intracellular localization and widespread contacts of the enzyme with cytosol.     

Identification of chemosensory sensilla activating antennular grooming behavior in the Caribbean spiny lobster,Panulirus argus

Crustaceans such as crabs and lobsters clean or 'groom' their olfactory organ, the antennule, by wiping it through a pair of mouthpart appendages, the third maxillipeds. In the lobster, only a few chemicals found in prey extracts, especially glutamate, elicit grooming. Chemosensory input driving grooming is likely to be mediated via sensilla located on antennules and third maxillipeds. Chemosensory antennular sensilla are innervated by neurons with central projections either to the glomerular olfactory lobe (aesthetasc sensilla) or to non-glomerular antennular neuropils (nonaesthetasc sensilla). By selectively ablating the chemosensory sensilla on the antennules and the third maxillipeds we have determined that the aesthetascs are necessary and sufficient to drive grooming behavior. Chemosensory activation of antennular grooming behavior likely follows a 'labeled-line' model in that aesthetasc neurons tuned to glutamate provide adequate input via the olfactory lobe to motor centers in the brain controlling antennular movements.     

Comparative study of the olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius)

Summary The olfactory epithelium of the three-spined stickleback (Gasterosteus aculeatus) and the nine-spined stickleback (Pungitius pungitius) has been studied with a conventional histochemical and a novel immunological staining technique. In both species, the sensory epithelium is arranged in folds separated by non-sensory epithelial tissue. In the nine-spined stickleback, intrinsic folds consisting of non-sensory cells are found in the apical part of the sensory epithelium where they divide the surface of the sensory epithelium into small islets. These non-sensory cells are non-ciliated, flattened and piled on top of each other; they contain numerous electron-translucent vesicles. The intrinsic folds are absent from the sensory epithelium of the three-spined stickleback. In both species, axons of receptor cells form a layer of fibers in the sensory epithelium immediately above the basal cells. In the three-spined stickleback, thick branches of the olfactory nerve are frequently found in this layer. These branches are only occasionally observed in the sensory epithelium of the nine-spined stickleback. Thus, the three-spined stickleback and the nine-spined stickleback show considerable differences in the organization of the sensory regions of the olfactory epithelium.     

Cholinesterases in peripheral nervous system. III. Validity of localisation around the node of Ranvier

Synopsis A series of experiments was designed to exclude the possibility that the acetylcholinesterase reaction of the rat's node of Ranvier is artefactual. Copper ions were not bound by perinodal acid mucopolysaccharides (or axons) at pH 6.0, which is the same value as that used in the histochemical cholinesterase techniques. No positive evidence was found for diffusion of acetylcholinesterase out of teased nerve fibres. Likewise, no evidence was obtained that acetylcholinesterase, acetylthiocholine or thiocholine are bound at pH 6.0 by perinodal acid mucopolysaccharides (or axons). It is concluded that local non-specific binding of copper, enzyme, substrate or reaction-product cannot account for the acetylcholinesterase reaction in either the node of Ranvier or the axon.     

Ocean acidification alters foraging behaviour in Dungeness crab through impairment of the olfactory pathway

Crustacean olfaction is fundamental to most aspects of living and communicating in aquatic environments and more broadly, for individual- and population-level success. Accelerated ocean acidification from elevated CO₂ threatens the ability of crabs to detect and respond to important olfactory-related cues. Here, we demonstrate that the ecologically and economically important Dungeness crab (Metacarcinus magister) exhibits reduced olfactory-related antennular flicking responses to a food cue when exposed to near-future CO₂ levels, adding to the growing body of evidence of impaired crab behaviour. Underlying this altered behaviour, we find that crabs have lower olfactory nerve sensitivities (twofold reduction in antennular nerve activity) in response to a food cue when exposed to elevated CO₂. This suggests that near-future CO₂ levels will impact the threshold of detection of food by crabs. We also show that lower olfactory nerve sensitivity in elevated CO₂ is accompanied by a decrease in the olfactory sensory neuron (OSN) expression of a principal chemosensory receptor protein, ionotropic receptor 25a (IR25a) which is fundamental for odorant coding and olfactory signalling cascades. The OSNs also exhibit morphological changes in the form of decreased surface areas of their somata. This study provides the first evidence of the effects of high CO₂ levels at multiple levels of biological organization in marine crabs, linking physiological and cellular changes with whole animal behavioural responses.     

Reciprocal interactions between olfactory receptor axons and olfactory nerve glia cultured from the developing moth Manduca sexta

In olfactory systems, neuron-glia interactions have been implicated in the growth and guidance of olfactory receptor axons. In the moth Manduca sexta, developing olfactory receptor axons encounter several types of glia as they grow into the brain. Antennal nerve glia are born in the periphery and enwrap bundles of olfactory receptor axons in the antennal nerve. Although their peripheral origin and relationship with axon bundles suggest that they share features with mammalian olfactory ensheathing cells, the developmental roles of antennal nerve glia remain elusive. When cocultured with antennal nerve glial cells, olfactory receptor growth cones readily advance along glial processes without displaying prolonged changes in morphology. In turn, olfactory receptor axons induce antennal nerve glial cells to form multicellular arrays through proliferation and process extension. In contrast to antennal nerve glia, centrally derived glial cells from the axon sorting zone and antennal lobe never form arrays in vitro, and growth-cone glial-cell encounters with these cells halt axon elongation and cause permanent elaborations in growth cone morphology. We propose that antennal nerve glia play roles similar to olfactory ensheathing cells in supporting axon elongation, yet differ in their capacity to influence axon guidance, sorting, and targeting, roles that could be played by central olfactory glia in Manduca.     

A comparison of the localization of acetylcholinesterase in the rat brain as demonstrated by enzyme histochemistry and immunohistochemistry

Summary The localization of acetylcholinesterase (AChE) as revealed either by enzyme-histochemical or by immunohistochemical methods was compared in distinct regions of the rat brain. In general, the localization of AChE observed was nearly the same, whether revealed by histochemical demonstration of its catalytic activity or by immunohistochemical detection of the enzyme molecule itsclf, in all regions investigated. Penetration problems of the antibodies, however, arose on strong myelin sheaths of the facial nerve, for instance, where no immunohistochemical staining was found though there was a relatively strong histochemical reaction. These problems could be partly solved by increasing the normal concentration of Triton X-100 added to the immunohistochemical solutions (0.1%) to 2.5%. Furthermore, it seems that sites containing low amounts of AChE could be better detected by the enzymehistochemical method, whereas the depiction of structures (particularly of nerve fibres) was somewhat sharper with the immunohistochemical method.Dedicated to Professor Dr.T.H. Schiebler on the occasion of his 65th birthday     

Localization and activity of cholinesterases in Bidder's ganglion cells and nerve fibers of the frog heart

Summary The histochemical and cytochemical localization of cholinesterases in Bidder's ganglion of the frog heart was investigated and the activity of cholinesterases measured microgasometrically.When either acetylthiocholine or propionylthiocholine was used as substrate, the end product of histochemical and cytochemical reaction was found in all ganglion cells and nerve fibers, the only difference being in the amount of the accumulated precipitate. The end product was localized on the axolemma and in the smooth endoplasmic reticulum of nerve fibers; in the smooth and granulated endoplasmic reticulum of ganglion cells; in the synaptic region between the nerve ending and Schwann cells membrane; and, occasionally, in discontinous streches between the pre- and postsynaptic membrane. The nerve endings of the neurons of the Bidder's ganglion contain predominantly agranular vesicles with occasional clusters of granular ones.Neither the histochemical nor the cytochemical localization of enzyme activity could be detected when butyrylthiocholine was used as substrate. In microgasometric experiments splitting of butyrylcholine was hardly, if at all, measurable. Cholinesterases of the ganglion cells and of the nerve fibers of Bidder's ganglion hydrolyze propionylcholine but at a somewhat lower rate than acetylcholine.A part of this work has been presented at the VI. Congress of the Yugoslav Physiological Society, Ohrid, 1969.This work was supported by the Boris Kidri Foundation, Ljubljana, and by the Federal Research Council, Beograd.     

The relationship of acetylcholinesterase activity to optic fiber projections in the tiger salamander Ambystoma tigrinum

In the tiger salamnder the distribution of optic fibers, as revealed by the Fink-Heimer method, is compared with the localization of acetylcholinesterase, as revealed by histochemical methods. AChE activity coincides with optic nerve axons in the optic fiber layer of the retina, in the optic nerve, in the optic tracts and in every optic projection. Reginons of optic fiber terminals show heavier activity than optic fibers of passage. Comparison with other vertebrates is also made.     

Outgrowing olfactory axons contain the Reelin receptor VLDLR and navigate through the Reelin-rich cribriform mesenchyme

Chemosensory neurons in the olfactory epithelium (OE) project axonal processes to the olfactory bulb (OB) of the brain. During embryonic stages, on their trajectory to the OB, the outgrowing axons traverse the so-called cribriform mesenchyme, which is located between the OE and the OB. The molecular cues guiding these axons through the cribriform mesenchyme are largely unknown. To identify molecules influencing the axonal trajectory in the murine cribriform mesenchyme, we performed microarray analyses focusing on extracellular matrix (ECM) proteins present in this tissue. Thereby, the ECM protein Reelin turned out to be an interesting candidate. Reelin was found to be expressed by numerous cells in the cribriform mesenchyme during the embryonic stages when the first axons navigate from the OE to the OB. These cells were closely associated with olfactory axons and apparently lack glial and neuronal markers. In the mesenchyme underlying the OE, localization of the Reelin protein was not confined to the Reelin-expressing cells, but it was also observed to be widely distributed in the ECM—most prominently in regions traversed by olfactory axons. Importantly, these axons were found to be endowed with the Reelin receptor very-low-density lipoprotein receptor (VLDLR). Finally, Reelin expression was also detectable in neuronal cells of the OB, which are contacted by VLDLR-positive olfactory axons. In summary, the results of the present study suggest that a Reelin/VLDLR signaling pathway might contribute to the formation of olfactory projections to the OB and the establishment of initial contacts between the incoming axons and neurons in the OB. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Funding:  This work was supported by the Deutsche Forschungsgemeinschaft.     

The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio

We have examined neuronal differentiation and the formation of axon tracts in the embryonic forebrain and midbrain of the zebrafish, between 1 and 2 days postfertilisation. Axons were visualised with three techniques; immunocytochemistry (using HNK-1 and antiacetylated tubulin antibodies) and horseradish peroxidase (HRP) labelling in whole-mounted brains, and transmission electron microscopy. Differentiation was monitored by histochemical staining for acetylcholinesterase (AChE). These independent methods demonstrated that a simple grid of tracts and commissures forms the initial axon scaffold of the brain. At 1 day, the olfactory nerve, four commissures, their associated tracts and three other non-commissural tracts are present. By 2 days, these tracts and commissures have all greatly enlarged and, in addition, the optic nerve and tract, and three new commissures and their associated tracts have been added. Small applications of HRP at various sites revealed the origins and projections of some of these earliest axons. Retrogradely labelled cell bodies originated from regions that were also positive for AChE activity. At 1 day, HRP-labelled axons were traced: (1) from the olfactory placode through the olfactory nerve to the dorsal telencephalon; (2) from the telencephalon into the tract of the anterior commissure and also to the postoptic region of the diencephalon; (3) from the hindbrain through the ventral midbrain and diencephalon to the postoptic commissure; (4) from the dorsal diencephalon (in or near the epiphysis) to the tract of the postoptic commissure; (5) from ventral and rostral midbrain through the posterior commissure. Three new projections were demonstrated at 2 days: (1) from the retina through the tract of the postoptic commissure to the tectum; (2) from the telencephalon to the contralateral diencephalon; and (3) from the telencephalon to the ventral flexure. These results show that at 1 day, the zebrafish brain is impressively simple, with a few small, well-separated tracts but by 2 days the brain is already considerably more complex. Most of the additional axons added onto pre-existent tracts rather than pioneered new ones supporting the notion that other axons play a crucial role in the guidance of early central nervous system (CNS) axons.     

Cell surface carbohydrates reveal heterogeneity in olfactory receptor cell axons in the mouse

Cell surface carbohydrates, both in the olfactory system and elsewhere, have been proposed to play critical roles in axon guidance and targeting. Recent studies have used plant lectins to study the heterogeneous distribution of carbohydrates in the olfactory system. One lectin, Dolichos biflorus agglutinin (DBA), heterogeneously labels subsets of glomeruli. In the olfactory epithelium DBA labeled a subset of olfactory sensory neurons (OSNs) including their cilia, dendrites, and somata. OSN axons were also labeled and readily observed in the olfactory nerve and bulb. The patterns of glomerular innervation by DBA labeled (DBA(+)) axons were diverse; some glomeruli contained many labeled axons, while others contained few or no labeled axons. To characterize the heterogeneous innervation of glomeruli, we double labeled olfactory bulbs with DBA and an antibody to olfactory marker protein (OMP). OMP colocalized in most, but not all, DBA(+) axons. To determine if those axons that did not express OMP were immature, we double labeled olfactory bulbs with DBA and anti-GAP-43. GAP-43 rarely colocalized with DBA, suggesting that DBA(+) axons are not, as a population, immature. Triple labeling with all three markers revealed a small subset of DBA(+) axons which did not express either OMP or GAP-43. Electron microscopy established that DBA labels axons in the olfactory nerve and DBA-labeled axons form typical glomerular axodendritic synapses.