C‐bouton components on rat extensor digitorum longus motoneurons are resistant to chronic functional overload

Abstract Mammalian motor systems adapt to the demands of their environment. For example, muscle fibre types change in response to increased load or endurance demands. However, for adaptations to be effective, motoneurons must adapt such that their properties match those of the innervated muscle fibres. We used a rat model of chronic functional overload to assess adaptations to both motoneuron size and a key modulatory synapse responsible for amplification of motor output, C‐boutons. Overload of extensor digitorum longus (EDL) muscles was induced by removal of their synergists, tibialis anterior muscles. Following 21 days survival, EDL muscles showed an increase in fatigue resistance and a decrease in force output, indicating a shift to a slower phenotype. These changes were reflected by a decrease in motoneuron size. However, C‐bouton complexes remained largely unaffected by overload. The C‐boutons themselves, quantified by expression of vesicular acetylcholine transporter, were similar in size and density in the control and overload conditions. Expression of the post‐synaptic voltage‐gated potassium channel (KV2.1) was also unchanged. Small conductance calcium‐activated potassium channels (SK3) were expressed in most EDL motoneurons, despite this being an almost exclusively fast motor pool. Overload induced a decrease in the proportion of SK3+ cells, however, there was no change in density or size of clusters. We propose that reductions in motoneuron size may promote early recruitment of EDL motoneurons, but that C‐bouton plasticity is not necessary to increase the force output required in response to muscle overload.

activation patterns, which drives targeted, functional adaptation of skeletal muscle, altering metabolic signalling and expanding the vascular bed (Jensen et al., 2004). Additionally, chronic increases in loading drive muscle hypertrophy and increase force capacity (Mitchell et al., 2012). Moreover, constitutive fibre types, usually classified as slow oxidative (S-Type I), fast fatigue resistant (FR-Type IIA) and fast fatigable (FF-Type IIB/X), can undergo adaptive changes that alter muscle functional properties. For example, endurance training increases the proportion of Type I and IIA oxidative fibres resulting in improved fatigue resistance (Green et al., 1983).
Motoneurons innervating skeletal muscle also adapt to changing demands with electrophysiological adaptations matched to properties of the innervated muscle. This was elegantly shown by crossreinnervation studies in which forced mismatches between muscle and motoneuron properties were induced by surgically re-routing motor axons from one muscle to another (e.g. medial gastrocnemius [MG] to soleus; Dum et al., 1985;Foehring et al., 1987). Following recovery, assessments of motoneuron properties at a chronic stage showed a shift towards those of the reinnervated muscle, suggesting mechanisms of plasticity exist within the motor unit to match muscle and motoneuron properties.
Physiological stimuli such as exercise also induce functional adaptations in motoneuron properties: endurance training in rats induces increases in the motoneuron medium after hyperpolarisation (mAHP) amplitude, consistent with a shift to a more fatigue-resistant phenotype (Gardiner et al., 2006). Thus, physiological adaptations in motor units should always be reflected centrally and peripherally.
Altered neuromuscular demand can also stimulate changes in organisation of synaptic inputs to motoneurons and in expression of post-synaptic membrane proteins, both of which reflect changes in neuromuscular activity (Arbat-Plana et al., 2017;Woodrow et al., 2013). Thus, adaptations in spinal cord physiology parallel changes in the muscular system. However, it is less clear what type of inputs are sensitive to chronic changes in activity, and the degree to which they might adapt. For example, gain-of-function adaptations in neuromodulatory inputs that amplify motor output may be a beneficial response to chronic increases in load. Equally, neuromodulatory systems may already be equipped to respond to chronic increases in load and thus do not need to adapt the pre-or post-synaptic molecular machinery.
Motoneurons receive neuromodulatory cholinergic synapses, termed C-boutons, because of their association with post-synaptic subsurface cisternae (SSC; Conradi, 1969) that regulate the mAHP (Miles et al., 2007). These somatic/proximal dendritic synapses can amplify motor output in a task-specific manner. The system is comprised of V0 C premotor interneurons (Zagoraiou et al., 2009), C-bouton synapses, and clusters of several different post-synaptic membrane proteins (Witts et al., 2014). Recent work has suggested mechanisms for how some of these components may contribute to amplification of motor output during high force output tasks such as swimming (Nascimento et al., 2020;Romer et al., 2019;Soulard et al., 2020). Activation of type 2 muscarinic acetylcholine receptors (m2AChR) on spinal motoneurons results in a reduction in the mAHP amplitude and an increase in excitability as measured by the frequency/current (ƒ−I) relationship (Miles et al., 2007). The mAHP current is carried by small conductance, calcium-activated potassium channels SK2 & SK3, which are differentially expressed in fast and slow motoneurons, endowing them with their respective mAHP characteristics (Deardorff et al., 2013).
Also clustered opposite C-boutons are delayed rectifier, voltagegated potassium channels, K V 2.1 (Muennich & Fyffe, 2004). The role of these channels is less well understood, but recent evidence suggests they may act as 'molecular rheostats', capable of maintaining firing during high synaptic drive or supressing firing to protect motoneurons from excitotoxicity (Romer et al., 2019). It has been shown that m2AChRs modulate K V 2.1 channels by reducing action potential half-widths and increasing the inter-spike AHP, which aids recovery of Na + channels during high synaptic drive; thus, supporting high-frequency firing (Nascimento et al., 2020). Furthermore, as in various brain regions (Murakoshi et al., 1997;Park et al., 2006), the distribution of motoneuron K V 2.1 channels is plastic, suggesting they may play a role in neuromuscular adaptation in health and disease . Specifically, high activity states that increase intracellular calcium concentrations cause K V 2.1 channels to rapidly de-cluster, which lowers their activation threshold and increases conductance. In lower activity states, Kv2.1 channels coalesce into macro-clusters that form physical links with the SCC (Deardorff et al., 2021), and are thought to be non-conducting.
However, the functional significance of K V 2.1 channel conducting and non-conducting roles in behaviour has yet to be determined.
Several groups have studied plasticity of C-boutons in disease states. For example, Landoni et al. (2019) showed that C-bouton transmission initially compensates for progression of motor deficits during motoneuron loss in SOD1 amyotrophic lateral sclerosis (ALS) mice. Conversely, Konsolaki et al. (2020) have shown that C-bouton inactivation improves motor performance but not survival in SOD1 ALS mice. It is difficult, however, to separate mechanisms associated with disease or injury and chronic physiological overload of the neuromuscular system. Therefore, it is important to study how chronic changes in neuromuscular demand affect central components of the motor system, such as the motoneuron and its modulatory inputs.
Here, we asked whether such changes in muscular demand lead to corresponding adaptations in motoneurons and at C-bouton synapses. We used a model of chronic neuromuscular overload, as similar models have previously been shown to induce central and peripheral adaptations in motor units (Chalmers et al., 1991;Ianuzzo et al., 1976;Krutki et al., 2015;Rosenblatt & Parry, 1992). This involved extirpating the tibialis anterior (TA) muscle to increase loading of the remaining synergist extensor digitorum longus (EDL) muscle in adult rats for 21 days. We confirmed effectiveness of the overload stimulus by assessing changes in muscle physiology, showing a shift to a more fatigue-resistant phenotype. We then studied EDL motoneuron adaptations using retrograde tracers, and showed a corresponding reduction in cross-sectional area. Although we hypothesised that overload would induce adaptations to C-bouton organisation that correlated with adaptations seen in muscle physiology, there were no measurable differences in sizes and densities of both pre-(C-boutons) and post-synaptic (K V 2.1 & SK3) components, however, there was a reduction in the proportion of SK3 + cells following overload. Our results show that in conjunction with a slower muscle phenotype following overload, there is a corresponding decrease in motoneuron size. We suggest that this central adaptation may compensate for increased functional demands by reducing motoneuron rheobase and increasing excitability. Furthermore, anatomical plasticity of the neuromodulatory C-bouton complex is not necessary to produce increased force output in this model of chronic functional overload.

| Animals
Male Wistar rats (N = 14; 283 ± 29 g) were housed under a 12:12 light-dark cycle in a temperature-controlled 21°C environment, with ad libitum access to food and water. Animals were randomly allocated to either control of overload conditions.

| Animal surgical procedures
All animal surgeries were completed by competent Home Officeapproved PIL holders, under aseptic conditions. Surgical anaesthesia was induced and maintained with isoflurane (5% and 2%, respectively, in 100% O 2; IsoFlo ® ; Zoetis UK Ltd).

| Muscle overload
An incision was made two thirds up the length of the right TA towards the lateral side of the muscle. The covering fascia was cleared exposing the TA muscle, enabling sectioning of the distal tendon above the retinaculum and as close as possible to the proximal insertion (Egginton et al., 2011). Upon releasing the tendons, the TA was bluntly dissected from the lateral tibia surface and removed, taking care not to damage the underlying EDL. Skin was closed with 5-0 Mersilk suture (Ethicon, Johnson & Johnson Medical Ltd). Animals received subcutaneous analgesia (0.015 mg/kg, Vetagesic ® ; Ceva) and antibiotic (2.5 mg/kg, Baytril ® ; Bayer) for 2 days post-surgery.
Removal of the TA muscle increases the load burden on its synergist, the EDL. We, thus, use the term "overload" for this condition.
The overload period lasted 21 days.

| Motoneuron tracing
Retrograde fluorescent tracers were injected into the EDL 5 days prior to terminal experiments. Tracers were injected into the medial and lateral compartments of the EDL for separate assessment of motoneurons innervating these compartments (data not included). One μL of 1.5% 647 nm CTβ Alexa Fluor ™ Conjugate (Invitrogen) was injected into both medial and lateral compartments; 3 μL of 1.5% Fast Blue (Polyscience, Inc.) was injected only into the medial compartment. Skin was closed using 5-0 Mersilk suture (Ethicon, Johnson & Johnson Medical Ltd). Animals received analgesic (0.015 mg/kg, Vetagesic ® ; Ceva) and antibiotic (2.5 mg/kg, Baytril ® ; Bayer) subcutaneously for 2 days post-surgery.

| In situ muscle fatigability
Anaesthesia was induced with isoflurane (4% in 100% O 2 ) and maintained by constant infusion (30-35 mg kg −1 h −1 ) of alfaxalone (Alfaxan: Jurox) via a catheter implanted into the external jugular vein. A tracheotomy was performed to facilitate spontaneous breathing. Blood pressure and heart rate were monitored in LabChart 8 (AD Instruments) via a carotid artery catheter connected to a pressure transducer (AD Instruments).
EDL twitch force was quantified using a lever arm transducer system (305B-LR; Aurora Scientific) and LabChart 8 (AD Instruments).
Unimpeded access to the EDL was enabled by dissection of surrounding fascia, the distal tendon was then cut and attached to the lever arm of the force transducer. The peroneal nerve was exposed and indirectly stimulated using bipolar stainless-steel electrodes (Hudlicka et al., 1977), with muscle length and electrical current delivery optimised to generate maximal isometric twitch force. Fatigue resistance of the EDL was determined using a protocol (10 Hz electrical stimulation, 0.3 ms pulse width) to elicit a series of isometric contractions over 3 min. A fatigue index (FI) was calculated as the ratio of end-stimulation tension to peak tension (FI = end-stimulation tension/peak tension), using the mean of 5 consecutive twitches.

| Tissue preparation
Following successful in situ recordings, and remaining under anaesthesia, EDL were dissected and the muscle mid-belly was snap frozen in liquid nitrogen cooled isopentane for muscle capillary analysis. All frozen muscle tissue was stored at −80°C until cryosectioning. Next, animals were transcardially perfused with 0.1 M phosphate buffer and fixed with 4% paraformaldehyde. Spinal columns were removed immediately after perfusion and post-fixed in 4% PFA for 24 h. Spinal cords were carefully dissected and cryoprotected in 30% sucrose at 4°C for 7 days. Next, the lumbar segments were isolated, frozen in OCT (Agar Scientific) and stored at −20°C.
Indices for capillary-to-fibre ratio (C:F) and capillary density (CD) were derived from histological sections. These global indices describe gross changes in capillary supply, however, they lack descriptive power of local capillary distribution. The local capillary supply is a critical determinant of functional capacity and of significant importance in the functional overload model which presents with a significant angiogenic response and fibre hypertrophy (Kissane et al., 2020;Tickle et al., 2020). Therefore, to investigate the influence of concurrent expansion of the capillary bed and fibre hypertrophy on muscle function, we mathematically modelled skeletal muscle oxygen transport kinetics (Al-Shammari et al., 2019). Briefly, capillary distributions were digitally derived from histological sections and used to model as a point source of O 2 and estimations of tissue PO 2 were predicted using a number of model assumptions: oxygen demand (15.7 × 10 −5 ml O 2 ml −1 s −1 ), myoglobin concentration (10.2 × 10 −3 O 2 ml −1 ), oxygen solubility (3.89 × 10 −5 ml O 2 ml −1 mmHg −1 ), myoglobin diffusivity (1.73 × 10 −7 cm 2 s −1 ) and capillary radius (1.8-2.5 × 10 −4 cm; Al-Shammari et al., 2019).
We performed histological assessments of the fibre-type distributions in both conditions, but tracer loading of the muscle reduced sample quality. These data were, therefore, excluded.

| Spinal cord immunohistochemistry
Spinal cord immunohistochemistry was performed as previously described (Smith et al., 2017). In brief, L3-L6 segments were sectioned at 50 µm on a cryostat (−20°C) and free-floating sections were col-

| Confocal microscopy and quantitative analysis
Images were acquired with a Zeiss LSM 800 confocal microscope were created via surface rendering and thresholding. CTβ or Fast Blue was used to model the motoneuron surface. A masking feature was then used to select K V 2.1 or SK3 clusters contacting the motoneuron surface and/or proximal to the C-bouton. IMARIS was then used to generate volume and surface area data for each motoneuron, Cboutons, K V 2.1 clusters and SK3 clusters. To determine the motoneuron cell size, cross-sectional area through the centre of the nucleus was calculated using Image J (ImageJ, RRID:SCR_003070).
Data were then exported to Excel (Microsoft Excel, RRID:SCR_016137). Since all alpha-motoneurons contain C-boutons and K V 2.1, cells with no C-bouton or K V 2.1 surfaces were removed. C-bouton, SK3 and K V 2.1 channel densities were normalised to the motoneuron surface area. For statistical analyses, the mean synaptic and channel density for each motoneuron was the observational unit (n) and the average density per animal was the experimental unit (N).
Sample sizes were determined based on previous studies (Kissane et al., 2018). Shapiro-Wilks tests were performed to determine normality of the data, followed by either unpaired t-tests (normally distributed) or Mann-Whitney U tests (not normally distributed). Fisher's exact test was used to compare the proportion of cells expressing SK3. Data are presented throughout as mean ± standard deviation. All analyses were performed using Python scripts (RRID: SCR_008394) in the Jupyter notebooks environment (RRID: SCR_013995).

| Data Availability Statement
The data that support the findings of this study and a digital analysis notebook are openly available in zenodo at https://zenodo.org/ badge/ lates tdoi/33632 7574 reference number [RRID:SCR_002630].

| Chronic functional overload induces functional shift to slower EDL phenotype
Previous studies of EDL muscle overload by removal of the TA synergist have shown an anatomic and physiologic shift to a slower phenotype (Rosenblatt & Parry, 1992;Rosenblatt & Parry, 1993). We were not able to reliably analyse fibre-type distribution in this study due to the loading of muscles with neuro-anatomical tracers. However, muscle weight was significantly greater in the overload condition, suggesting hypertrophy of EDL fibres (Control = 0.06 ± 0.01 g, N = 5 vs. Overload 0.09 ± 0.02 g, N = 7, p = 0.001).

| Chronic functional overload improves fatigability in EDL muscles
In order to determine if there were physiological adaptations in EDL muscles following overload, we assessed muscle twitch force and fatigability using an in vivo anaesthetised preparation.
Overall, our physiological data confirm a shift to a slower, more fatigue-resistant phenotype following overload.

| Chronic functional overload reduces EDL motoneuron soma cross-section area
Motoneuron properties are matched to the muscle fibre types they innervate. Motoneurons innervating Type 1 muscle fibres are smaller than those innervating IIa, which are smaller than those innervating IIb/IIx (Burke, 1967). In the overload model, we found a shift in the distribution of motoneuron sizes towards smaller motoneurons, leading to an overall reduction in EDL motoneuron size following removal of TA (Control = 174 ± 248, N = 6, n = 285 vs.

| Overload has no effect on C-bouton innervation of EDL motoneurons
C-bouton synapses are terminals of the V0 C interneuron circuit responsible for task-specific amplification of motor output (Miles et al., 2007;Zagoraiou et al., 2009). Because the overload condition removed the contribution of the synergist, TA, to locomotion and necessitated increased force output from the EDL, we asked whether an increase in C-bouton synapses (Figure 3a) occurs in order to meet the increased demands placed on EDL motoneurons. We thus assessed the effect of overload on the density and size of C-bouton inputs. vs. Overload = 23.3 ± 6.9 µm 2 , N = 5, n = 259, p = 0.48, Figure 3c,c1).
That is, there were no discernible changes to the presynaptic component of C-bouton inputs to EDL motoneurons.

| K V 2.1 channel density and area are unaffected by overload
Although we saw no change in presynaptic C-bouton characteristics, changes in the post-synaptic protein complex could alter synapse function. K V 2.1 channels are thought to be important for facilitating high-frequency motoneuron firing and are recruited for C-bouton amplification of motor output (Nascimento et al., 2020). In addition to the large clusters found at the C-bouton, K V 2.1 channels are found in smaller clusters distributed throughout the membrane

| SK3 channels are expressed in most EDL motoneurons but are unaltered by chronic functional overload
In addition to K V 2.1 channels, small conductance potassium channels (SK) are also clustered on the motoneuron post-synaptic membrane opposing C-boutons. SK2 and SK3 channels are responsible for the calcium-dependent potassium currents underlying the mAHP and, therefore, regulate motoneuron firing frequencies.
Previous work has suggested that SK2 channels are expressed in all motoneurons, whereas in the specific pools studied (mainly tibial motoneurons), SK3 channels are selectively expresssed in  N) and statistical tests are as follows: (b, c), control N = 6, overload N = 5, unpaired t-tests. Scale bar = 10 µm. Whiskers extend to 1.5 × SD of the mean. EDL, extensor digitorum longus slow motoneurons (Deardorff et al., 2013;Dukkipati et al., 2018).
Although the EDL muscle is mainly comprised of fast fatiguable (FF) units, there are also fast FR and a small proportion of type I slow (S) units (Kissane et al., 2018). We reasoned that, given the shift to a more fatigue-resistant phenotype following chronic overload (Figure 1) (d-h), control N = 6, overload N = 5. Statistical tests performed were as follows: (e, g, and i), unpaired t-tests; (f and h) Mann Whitney U tests. *Represents a statistically significant difference (p < 0.05). Scale bars in (a-d) = 10 µm, (a1-d1) = 2.5 μm. Whiskers extend to 1.5 × SD of the mean

| DISCUSS ION
The results presented here demonstrate that chronic overload of the rat EDL muscle induces significant adadptations to muscle fibre capiliary innervation and contractile properties. Specifically, 21 days functional overload resulted in an increase in EDL capiliary-to-fibre ratio and fatigue resistance, paralleled by a decrease in twitch and tetanic force production. We reasoned that this shift to a slower phenotype in the overloaded EDL muscle may be reflected anatomically in their motoneuron characteristics. This was confirmed by a decrease in motoneuron soma cross-sectional area. We also hypothesised that a key spinal neuromodualtory input, C-boutons, would change so as to compensate for the increased neuromuscular demand during overload. However, while we did find a decrease in the proportion of SK3 + neurons, we were unable to detect significant adaptations in size or density of key components of the C-bouton complex (C-bouton synapse, SK3 and K V 2.1 channels). Importantly, we also show that SK3, a suggested molecular marker for slow motoneurons (Dukkipati et al., 2018), was expressed in most EDL motoneurons, despite these being mainly fast type (Kissane et al., 2018). The overload-induced shift to a more aerobic phenotype in EDL muscle was reflected in the central portion of the motor unit, by a shift to smaller sizes for motoneurons in this condition. Motoneuron size is inversely related to input resistance and positively correlates with rheobase (Henneman, 1957), meaning changes in motoneuron size may lead to altered recruitment thresholds across the motor pool. Previous electrophysiological assessments of overloaded rat MG motoneurons demonstrated significant reductions in rheobase, associated with increased input resistance and a leftward shift in the frequency current (ƒ−I) relationship in FF motoneurons, indicating that less input was required to activate these motoneuorns (Krutki et al., 2015). Taken together, these changes in motoneuron properties would be matched to peripheral changes and mean that the probability of recruitment and firing rate is increased for a given synaptic drive in the overload compared to the control condition.

| C-bouton complex is largely unaltered by chronic functional overload
The C-bouton is a neuromodulatory synapse responsible for taskspecific amplification of motor output, and is anatomically characterised by a dense aggregation of proteins at the opposing post-synaptic membrane (Conradi, 1969;Deardorff et al., 2014). C-bouton modulation increases the ƒ-I slope of motoneurons through activation of m2AChRs and subsequent reductions in the amplitude and duration of the mAHP, mediated by SK channels (Miles et al., 2007). C-bouton SK2 channels are expressed in almost all motoneurons regardless of type, whereas previous work in extensor motoneurons shows that SK3 channels are preferentially found on S type motoneurons and thus are likely responsible for larger mAHP conductances (Deardorff et al., 2013;Dukkipati et al., 2018). Surprisingly, we found that most EDL motoneurons expressed large SK3 clusters to some degree.
Thus, in the rat EDL motor pool, it would seem that SK3 expression cannot be reliably used as a binary molecular marker to identify slow motoneurons.
There was a decrease in the proportion of SK3 + motoneurons in the overload condition. Given prior evidence that SK3 channels are associated with longer AHPs and slower phenotypes (Deardorff et al., 2013), the "loss" of SK3 expression could be considered to indicate a shift to a faster phenotype-a finding in contrast to our other findings. However, considering that we did not detect a decrease in SK3 expression (cluster size or density) in the SK3-positive cells, it is possible that the motoneurons that presumably 'lost' SK3 clusters had lower expression levels before overload. Taken together, it is not clear whether the reduction in the number of SK3 + neurons had significant functional implications.
We found no effect of overload on the size or density of C-boutons, K V 2.1 channels or SK3 channels on EDL motoneurons, suggesting that both pre-and post-synaptic components of the synaptic complex are largely unaffected by a stimulus which induced changes in muscle fibres and motoneuron size. This was unexpected, especially for SK3, as the motoneuron mAHP has been shown to be increased following chronic overload (Krutki et al., 2015). It is possible that there were changes in other SK isoforms, but given the propensity of SK3 channels to be differentially expressed in motoneuron types, we focused on them.
There are several potential reasons why we did not detect adaptations in C-bouton complexes. Firstly, certain motoneuron properties, such as C-bouton synapses, might be somewhat resistant to plasticity (Chalmers et al., 1991). Secondly, C-bouton synapses may already be organised to meet the increased demand following overload, and so even if they were more active, there may be no need for anatomical adaptation. Thirdly, C-bouton innervation may be sensitive to certain types of stimuli, but overload is either not appropriate or sufficent to stimulate plasticity. In this vein, K V 2.1 organisation on the motoneuronal membrane seems to be affected mainly by extremely high or pathological levels of activity (Romer et al., 2019;Romer et al., 2014), whereas our overload model induces a physiological load increase. Moreover, it is possible that K V 2.1 clustering was modulated during the early phases of overload, when the stimulus is greatest, and returned to 'normal' once motor adaptation was complete. Furthermore, we now know that K V 2.1 channels make physical links with the SSC via vesicle associated membrane protein (VAMP)-associated proteins (VAPs; Deardorff et al., 2021;Johnson et al., 2018;Kirmiz et al., 2018). Although the physiological relevance of this structural role is unknown, evidence from brain neurons suggests that the K V 2.1-VAP interaction maintains tight plasma membrane-SSC junctions (Johnson et al., 2018). Because many of the proteins located at the C-bouton are Ca 2+ modulated, and the region of the SSC functions as a local Ca 2+ microdomain, C-bouton activity could affect associated protein function (SK, K V 2.1 etc) by modulating Ca 2+ flux in the microdomain (Deardorff et al., 2021).
Thus, dispersal of K V 2.1 would be counterproductive in response to overload, as it would theoretically limit C-bouton function. However, it is important to note that the molecular mechanisms downstream of C-bouton activation of m2AChRs have yet to be described.
In conclusion, our results show that a shift to a slower phenotype in EDL muscles is accompanied by a reduction in EDL motoneuron size, which perhaps allows EDL motor units to be recruited with less synaptic drive. The C-bouton complex, a key neuromodulatory synapse, is, however, anatomically unaffected by overload, suggesting that adaptation of these synapses was not neccessary. Whether Cbouton function adapts in other environmental conditions remains to be seen.