Locomotor activities as a way of inducing neuroplasticity: insights from conventional approaches and perspectives on eccentric exercises

Corticospinal excitability, and particularly the balance between cortical inhibitory and excitatory processes (assessed in a muscle using single and paired-pulse transcranial magnetic stimulation), are affected by neurodegenerative pathologies or following a stroke. This review describes how locomotor exercises may counterbalance these neuroplastic alterations, either when performed under its conventional form (e.g., walking or cycling) or when comprising eccentric (i.e., active lengthening) muscle contractions. Non-fatiguing conventional locomotor exercise decreases intracortical inhibition and/or increases intracortical facilitation. These modifications notably seem to be a consequence of neurotrophic factors (e.g., brain-derived neurotrophic factor) resulting from the hemodynamic solicitation. Furthermore, it can be inferred from non-invasive brain and peripheral stimulation studies that repeated activation of neural networks can endogenously shape neuroplasticity. Such mechanisms could also occur following eccentric exercises (lengthening of the muscle), during which motor-related cortical potential (electroencephalography) is of greater magnitude and lasts longer than during concentric exercises (i.e., muscle shortening). As single-joint eccentric exercise decreased short- and long-interval intracortical inhibition and increased intracortical facilitation, locomotor eccentric exercise (e.g., downhill walking or eccentric cycling) may be even more potent by adding hemodynamic-related neuroplastic processes to endogenous processes. Besides, eccentric exercise is especially useful to develop relatively high force levels at low cardiorespiratory and perceived intensities, which can be a training goal alongside the induction of neuroplastic changes. Even though indirect evidence let us think that locomotor eccentric exercise could shape neuroplasticity in ways relevant to neurorehabilitation, its efficacy remains speculative. We provide future research directions on the neuroplastic effects and underlying mechanisms of locomotor exercise.


Introduction
During exercise, the primary motor cortex sends electrical impulses to trigger voluntary muscle contractions. The signal travels through nerves along the spinal cord (also termed corticospinal pathway), before reaching the alpha motoneuron, and then the muscle fibers it innervates. Corticospinal excitability, tested during an exercise or after its completion (during an isometric contraction or at rest) by applying transcranial magnetic stimulation (TMS) over the primary motor cortex, refers to "the efficacy of the corticospinal pathway to relay neural signals from higher brain areas to the muscle" (Weavil and Amann 2018). For stimulation intensities higher than the motor threshold, single-pulse TMS evokes a wave-like electrophysiological response in the targeted muscle, recorded by surface electromyography and termed motor evoked potential (MEP). MEP amplitude indicates Communicated by Michael Lindinger. the level of excitation of cortical neurons mono-or transsynaptically connected to spinal motoneurons (Groppa et al. 2012). During voluntary contraction (often isometric), the MEP is followed by the absence of muscle activity -silent period-, that mirrors the duration of inhibitions located at the cortical and spinal levels (Škarabot et al. 2019b;Yacyshyn et al. 2016). Paired-pulse TMS techniques also provide evidence that the recruitment of cortical neurons is mediated by inhibitory and facilitatory processes interacting at the cortical level (for a review see Chen 2004). Particularly, the short-interval intracortical inhibition technique is thought to reflect the activity of gamma-aminobutyric acid A (GABA A ) inhibitory neurotransmitters, while the long-interval intracortical inhibition technique, as well as the silent period duration (when lasting more than 100 ms), would reflect the activity of GABA B inhibitors (Chen 2004). The intracortical facilitation technique informs on the activity of glutamatergic facilitatory networks (Chen 2004). By merging TMS to diffusion tractography or electroencephalography, previous studies reported that MEP amplitude was representative of the structural integrity of the corticospinal tract (Condliffe et al. 2019), while the duration of the silent period reflected the magnitude of the GABAergic neurotransmission (Farzan et al. 2013).
Across the lifespan, the central nervous system undergoes neuroplastic changes under the form of a selective strengthening or weakening of neural networks, through processes known as long-term potentiation or long-term depression, respectively (Dayan and Cohen 2011). Both accompanied by structural (e.g. synaptogenesis, neurogenesis, neural sprouting; Südhof and Malenka 2008) and functional changes (e.g. change in cortical map area or MEP amplitude; Kleim and Jones 2008), making TMS a possible tool to investigate cortical plasticity and the occurrence of neuroplasticity (Hallett 2007). In particular, changes in the balance between cortical inhibition and facilitation could be a determinant of ontogenetic development (Gu 2002), and is altered along with motor executive functions in individuals with neurodegenerative diseases (for a review see Vucic and Kiernan 2017) or recovering from a stroke (e.g. Dancause and Nudo 2011;Hummel et al. 2009). Interestingly, this balance was also modified with motor learning (Rosenkranz et al. 2007).
In this context, neurorehabilitation protocols using noninvasive stimulation techniques such as repetitive TMS or paired-associative stimulation have been developed in order to counteract deleterious neuroplasticity (Nitsche et al. 2012). Despite a growing interest for these methods over the past two decades, limitations such as their expensiveness and precautions of use in certain individuals (e.g., those with epilepsy or migraine) hinder their utilization in a wide population. Physical activity has thus been considered as a promising approach to modulate neuroplasticity in rehabilitation protocols. Those modulations depend on exercise characteristics, among which the type of muscle contraction. The latter is of primal importance as the neuroplastic effects of concentric (muscle shortening) and eccentric (muscle lengthening) exercises seem to be underlain by distinct mechanisms.
This review article focuses on locomotor exercises because they represent a non-negligible part of daily activities (e.g., walking cycling) and can be performed at a low energy cost. In addition, it is likely to trigger the release of certain neurotrophic circulating factors in greater quantity than resistance exercise, due to superior blood flow (Tsai et al. 2019), and may thus induce more pronounced neuroplastic changes. Locomotor exercises were shown to reduce low-back pain (Brumitt et al. 2013), to improve the quality of life of people with schizophrenia (Dauwan et al. 2016) and elderly (Fleg 2012), and to induce neuroplastic effects (Mellow et al. 2020) that are potentially beneficial to health (Vucic and Kiernan 2017), making of it a very convenient and versatile tool for rehabilitation. The present narrative review unfolds as follows: (1) the impact of conventional locomotor exercise on neuroplasticity assessed in non-exercised or exercised muscles; (2) likely underlying neuroplastic processes triggered in relation with the hemodynamic flow; (3) a detour by the non-invasive brain and peripheral stimulation studies to emphasize the existence of nervous mechanisms that endogenously result in neuroplastic changes; (4) a perspective as a consequence of the previous observations: eccentric exercise and more specifically locomotor exercise within this category, as a way to merge endogenous and hemodynamic-related neuroplastic mechanisms.

Physical exercise induces neuroplasticity
Physical exercise has consistently been reported as an efficient stimulus to promote neuroplasticity. Aerobic exercise notably reduces intracortical inhibition related to GABAergic concentration in a way similar to the leaning of a simple motor task (Floyer-Lea et al. 2006). This, among other phenomena such as an increase in the number of synapses in the motor cortex (Kleim and Jones 2008), could have accounted for improved motor skill retention in patients with chronic stroke (Nepveu et al. 2017) or Parkinson disease (Steib et al. 2018), when motor practice was implemented in addition to aerobic exercise.
It is nonetheless challenging to prescribe exercise for neuroplastic modulations to benefit patients, for at least five reasons: (1) corticospinal responsiveness differs between populations (e.g., corticospinal excitability is decreased and increased, in patients suffering from Huntington's and Alzheimer's diseases, respectively (Vucic et al. 2011). Certain neuroplastic modulations could thus be beneficial to some populations but detrimental to others; (2) a given exercise may induce distinct neuroplastic modulations in two pathological populations; (3) two facilitating paired-associative stimulation protocols applied successively had concurrent effects, depressing corticospinal excitability (Müller et al. 2007). These seem to be driven by homeostatic mechanisms, whereby the effects of physical exercise or non-invasive brain stimulation on neuroplasticity depend upon the effects induced by a precedent similar protocol (Abraham 2008). Performing an exercise could thus counterbalance the proexcitability effect of another; (4) in addition, inducing neuroplasticity is never the only focus of a physical exercise program; rather, the prescription must aim for a compromise between several targeted outcomes (e.g., decreasing cortical inhibition, strengthening lower-limb muscles, improving respiratory fitness), (5) finally, the influence of exercise characteristics (e.g., duration, intensity) on neuroplasticity remain unclear (Mellow et al. 2020).
Despite this last point, modulations of corticospinal excitability by exercise are not region-or muscle-specific and were reported in both exercised and remote (non-exercised) muscles.
Transient changes in the excitability of the corticospinal pathway have also been reported for muscles involved in the exercise, yet they seem to depend on the features of the exercise performed. In most studies, corticospinal excitability increased following submaximal single-joint exercise performed with the upper-or lower-limb (Kotan et al. 2015;Pitman and Semmler 2012;Williams et al. 2014). Nonetheless, similar exercises have led to unchanged (Finn et al. 2018), or depressed corticospinal excitability when exercise was carried-out until exhaustion (Brasil-Neto et al. 1993). Single-joint exercises have consistently depressed corticospinal excitability and increased silent period duration, when conducted at maximal intensity (e.g. Goodall et al. 2018;Kennedy et al. 2016).
Locomotor exercise, because it involves large muscle masses and leads to an important hemodynamic solicitation, has the potential to significantly modulate corticospinal excitability of exercised muscles (Sidhu et al. 2013). It was indeed found that both a maximal (Fernandez-del-Olmo et al. 2013) and submaximal (Jubeau et al. 2014;Temesi et al. 2013) cycling exercise (from 30-s to 80-min) can increase corticospinal excitability, assessed in exercised muscles. Findings are however very heterogeneous: corticospinal excitability was depressed at the end of an exercise at supra-maximal intensity, but unchanged at submaximal intensity (80% peak power output, Sidhu et al. 2012). Despite unchanged corticospinal excitability, short-interval intracortical inhibition either decreased immediately following self-selected low-intensity pedaling (Yamaguchi et al. 2012;Yamazaki et al. 2019), increased after exhaustive cycling at severe intensity-although the silent period was shorter-(92% peak oxygen uptake; O'Leary et al. 2016), or decreased after pedaling until exhaustion at moderate intensity (52% peak oxygen uptake; O' Leary et al. 2016).
Corticospinal excitability, assessed in a remote hand muscle was unchanged following cycling (Morris et al. 2019;Singh et al. 2014a;Smith et al. 2014;Walsh et al. 2019), but increased after running (Garnier et al. 2017). It thus seems that the mode of exercise-cycling vs running-might affect corticospinal excitability, yet more evidence is needed. All cycling studies, reported reduced short-interval intracortical inhibition (Singh et al. 2014a;Smith et al. 2014), and increased intracortical facilitation (Morris et al. 2019;Singh et al. 2014a) examined by paired-pulse TMS. Such modifications in the balance between cortical facilitation and inhibition for a remote muscle make the case that locomotor exercise is a promising strategy to modulate neuroplasticity for motor learning purposes.
As recently emphasized (Mellow et al. 2020), the diversity of experimental protocols makes it difficult to highlight any exercise characteristic primary influencing exerciseinduced neuroplasticity. For instance, an exercise causing significant fatigue typically diminishes corticospinal excitability by reducing motoneurons responsiveness and increasing inhibitory nociceptive afferent feedback to the brain (Gandevia 2001), masking the effects of other characteristics such as exercise intensity may have following a shorter exercise (i.e., too short to cause significant fatigue and feedback firing). It however seems that cardiorespiratory intensity is a key parameter that influences neuroplastic changes following locomotor exercise (see Fig. 1 for an overview of the effects of locomotor exercise intensity on resulting neuroplastic changes).

Exercise intensity affects hemodynamic-related processes underlying neuroplasticity
Mechanisms by which exercise triggers neuroplasticity may be linked with the increase in circulating neurotrophic factors (e.g. the brain-derived neurotrophic factor; BDNF) and hormones (e.g. insulin-growth factor 1) in the systemic circulation, known to enhance cellular stress resistance in the brain (van Praag et al. 2014). BDNF and Insulin-growth factor 1 is released in the systemic blood circulation in response to muscle contraction (Berg and Bang 2004;Matthews et al. 2009). BDNF can also be secreted directly by neurons in response to an increase in their activity, yet whether peripheral BDNF somehow passes the brain-blood barrier or if the brain produces all the BDNF concentrated in its tissues remains unclear (Marie et al. 2018).
Similar to corticospinal excitability modulations, the greatest increases in muscle BDNF levels were reported following high-intensity exercises (Knaepen et al. 2010). A likely explanation is that high-intensity exercise is accompanied by a proportional important blood flow and endothelial shear stress, responsible for BDNF release (Cefis et al. 2019). While high-intensity exercise could prompt neuroplasticity in healthy subjects, it can also increase circulating levels of cortisol (Rojas Vega et al. 2006), a hormone known to impair neuroplasticity (Sale et al. 2008) and hinder the effects of BDNF. This might explain why pedaling intensity was shown to have no influence on post-exercise corticospinal excitability of a remote hand muscle (McDonnell et al. 2013;Smith et al. 2014). Consequently, it seems that in order to promote neuroplasticity, exercise intensity should be high enough to increase BDNF levels, yet not too high to limit the release of cortisol. Even so, only high exercise intensities (80% of heart rate reserve) decreased shortinterval intracortical inhibition immediately after exercise cessation (Smith et al. 2014). Symptom-limited individuals are unable to exercise at a sufficient intensity to achieve a relatively high blood flow (Barak et al. 2017), but they seem to release significant amounts of BDNF at low-intensity levels (Knaepen et al. 2010). Even if correlations were found between concentrations in circulating factors following aerobic exercise and changes in corticospinal excitability (McDonnell et al. 2013), or functional outcomes such as an enhanced cognition are typically underlain by neuroplasticity (Nilsson et al. 2020), the causality relationship between the two remains somewhat elusive.
It is possible to induce neuroplastic changes directly via endogenous mechanisms (i.e., resulting from repeated activation of neural networks), at low cardiorespiratory intensities. The presence of such mechanisms is evidenced by non-invasive stimulation studies (see Sect. "Non-invasive stimulation studies highlight endogenous mechanisms of neuroplasticity"), and it may be possible to take advantage of them using eccentric exercise, which is already employed as a rehabilitation tool for other reasons (see Sect. "Eccentric exercise as an alternative to trigger endogenous neuroplastic processes").

Non-invasive stimulation studies highlight endogenous mechanisms of neuroplasticity
Moderate intensity pedaling has been shown to promote neuroplasticity when preceding non-invasive brain stimulation protocols. For example, the effects of paired-associative stimulation (Mang et al. 2014;Singh et al. 2014b) or thetaburst stimulation (McDonnell et al. 2013) on corticospinal excitability assessed in a remote hand muscle were enhanced when preceded by low (~ 60% predicted maximal heart rate) or moderate (65-70% predicted maximal heart rate) pedaling exercise. Other research groups demonstrated the influence muscle afferent feedback exerts on acute neuroplasticity, namely it increased corticospinal excitability after the application of peripheral electrical stimulation designed to imitate muscular contraction (Chipchase et al. 2011;Schabrun et al. 2012). Authors have proposed reduced cortical inhibition or unmasked silent synaptic connections to explain this modification (Chipchase et al. 2011). In addition, the connectivity between the primary sensory and the primary motor cortex was likely increased, due to afferent inputs elicited by the mixed influence of voluntary muscle contraction and electrical stimulation (Schabrun et al. 2012). On the other hand, protocols that elicited nociceptive sensory stimulation without voluntary contraction, depressed corticospinal excitability of the stimulated muscle (Chipchase et al. 2011;Mang et al. 2010;Schabrun et al. 2012), irrespective of stimulation frequency. In addition, Veniero and colleagues (2013) demonstrated that the increase in MEP amplitude observed following a facilitating paired-associative stimulation protocol was accompanied by a strengthened communication between the parietal and the motor cortical areas stimulated during the protocol.
Altogether, these results seem to indicate that locomotor exercise and non-invasive stimulation mainly trigger neuroplasticity via hemodynamic-related processes or repeated activation of exercise-related neural networks, respectively (see Fig. 2 for a synthesis of possible mechanisms triggering neuroplastic modulations following locomotor exercise). Even though combining the two methods allowed neuroplastic changes at moderate exercise intensities, the aforementioned drawbacks of stimulation techniques restrict the applicability of this approach. It is thus of greatest importance to find a readily implementable method providing similar benefits. Eccentric exercise (i.e., an active lengthening of the muscle), especially when locomotor, may prove efficient.

Eccentric exercise as an alternative to trigger endogenous neuroplastic processes
Eccentric exercise may be an alternative to conventional exercise, inducing neuroplasticity through endogenous mechanisms. It is known to elicit a lower cardiorespiratory demand (Abbott et al. 1952;Garnier et al. 2019;Lemire Fig. 2 Summary of the mechanisms (endogenous and/or hemodynamic-related) suggested to induce neuroplasticity after each type of locomotor exercise. Data related to conventional (i.e., concentric) and eccentric exercise are in blue and red font, respectively. Superscript numbers refer to the studies that provided the results featured below. Reference numbers: Neural Plast; 17: Berg and Bang (2004) (Clos et al. 2019;Elmer and Martin 2010) than conventional exercise at the same work rate. It has also been shown to induce limited muscle damage in pathological populations, such as individuals suffering from the chronic obstructive pulmonary disease (Pageaux et al. 2019;Vieira et al. 2011) or obesity (Julian et al. 2018;Thomazo et al. 2019), while exercising at highto-moderate force levels. Given the "challenging" brain control of eccentric contractions (Perrey 2018), such an exercise could foster neuroplasticity. Indeed, when executing eccentric contractions, the movement-related cortical potential, as assessed using electroencephalography, was of greater magnitude and started earlier before the movement (Fang et al. 2001(Fang et al. , 2004 than when performing concentric contractions. Other studies reported greater rises in blood-oxygenlevel-dependent (BOLD) signal in the primary sensory cortex (Yue et al. 2000) and in the supplementary motor area (Kwon and Park 2011) during wrist flexion movement, or in pre-frontal cortex during imagined eccentric than concentric elbow flexions (Olsson et al. 2012). Finally, near-infrared spectroscopy revealed a greater activation of the contralateral primary motor cortex during eccentric than concentric elbow flexions (Borot et al. 2018). These specific cortical activations before the onset of the movement were proposed to have a role in limiting the mechanical strain exerted on the muscle-tendon complex in order to preserve it from damage (Fang et al. 2004;Olsson et al. 2012).
As for conventional exercise, the features (e.g., volume, intensity) of eccentric exercise likely influence the way it modulates corticospinal excitability, notably whether the exercise involves a single joint or is locomotor.
Short-interval intracortical inhibition was lower during eccentric than concentric index finger abduction (Opie and Semmler 2016). Consistent findings also reported lower corticospinal excitability in eccentric compared with concentric single-joint contractions (Fang et al. 2004;Sekiguchi et al. 2003). Greater spinal inhibition, mediated by supraspinal mechanisms, was thus proposed to regulate the motor command, again to preserve the integrity of the muscle-tendon complex (Sekiguchi et al. 2001(Sekiguchi et al. , 2003. The mode of muscle contraction did not affect corticospinal excitability changes evaluated after elbow flexions (Latella et al. 2018;Löscher and Nordlund 2002) or knee extensions (Clos et al. 2020;Garnier et al. 2018). Some authors nevertheless reported reductions in short-interval intracortical inhibition (lasting 2 h, Pitman and Semmler 2012), long-interval intracortical inhibition and silent period duration (Škarabot et al. 2019a), and increases in intracortical facilitation (lasting 1 h, Latella et al. 2018). These changes were suggested to be the consequence of impaired motor control resulting from muscle damage (Pitman and Semmler 2012;Škarabot et al. 2019a). The long-lasting influence of eccentric contractions on cortical processes might also result from the complexity of the motor control required to perform these exercises-greater than for concentric contractions (Latella et al. 2018). This shift in the inhibition-excitation balance at the cortical level seems to exceed the brain hemisphere of the primary motor cortex controlling the active limb (i.e., the left hemisphere if the right limb is active). Magnetically stimulating the ipsilateral motor cortex during strong unilateral eccentric flexor carpi contractions, Howatson et al. (2011) found lower short-interval intracortical and inter-hemispheric inhibitions along with greater intra-cortical facilitation, than during same-torque concentric contractions. These phenomena may be amplified during a locomotor eccentric exercise as both limbs contract. From a rehabilitation point of view, the specific neural responses to eccentric contractions, related to the cross-education effect, could promote gains in the neuromuscular function of immobilized limbs. For instance, studies reported greater isometric and eccentric-but not concentric-strength gains in forearm flexors (Kidgell et al. 2015) and knee extensors (Hortobágyi et al. 1997) following an eccentric than concentric unilateral resistance training program with the contralateral homologous muscle. Using TMS pulses and surface EMG recordings, these authors suggested that eccentric strength training increases the excitability of the ipsilateral corticospinal tract (Kidgell et al. 2015) and enhances voluntary drive to untrained muscles (Hortobágyi et al. 1997).
Little is known about how the mode of muscle contraction affects neuroplastic changes following locomotor eccentric exercise (Fig. 1), which should combine a longer and more pronounced activation of motor and sensory cortical networks than its concentric counterpart (as shown in singlejoint exercises), with a low-but potentially significanthemodynamic solicitation. Despite this rationale, the model of muscle contraction does not seem to affect the global changes in corticospinal excitability measured in exercised lower limb or remote upper limb muscles, regardless of whether corticospinal excitability increased (Garnier et al. 2017 or remained unaffected (Walsh et al. 2019). Locomotor eccentric exercise may nevertheless have the potential to stimulate brain plasticity in a way partly similar to motor learning (Floyer-Lea et al. 2006;Rosenkranz et al. 2007). In fact, studies from our laboratory suggested that decline walking could specifically modulate the excitability of transcerebellar sensory pathway when associated with paired-associative stimulation (Garnier et al. 2017), and decrease short-interval intracortical inhibition assessed in an exercised muscle when implemented alone .
Furthermore, eccentric cycling, whose effects on neuroplasticity are mostly unknown (Clos et al. 2019;Walsh et al. 2019), is increasingly available in rehabilitation centers. This exercise modality allows those unable to walk due to joint pathologies or obesity to complete locomotor eccentric exercises. In addition to allowing force gains (Hoppeler 2016), and decreasing fat mass and increasing lean mass (Julian et al. 2018) while being well-tolerated in patients (LaStayo et al. 2013;Pageaux et al. 2019), eccentric cycling might enhance neuroplasticity and thus deserves its own set of investigations.

Research perspectives
The potential of locomotor exercises to shape neuroplasticity in ways beneficial to patients (Fig. 1) and several likely underlying mechanisms (Figs. 2) provide new research perspectives. First, studies are required to further describe the influence of conventional and locomotor eccentric exercise characteristics such as intensity, duration, induced-fatigue (related to training status), or movement frequency (e.g., pedaling cadence-Sidhu and Lauber 2020), to optimize clinical exercise protocols. Second, locomotor exerciseinduced neuroplasticity should be assessed along with functional outcomes (e.g., cognitive or motor task), which has seldom been the case thus far (Alibazi et al. 2020). Third, there is a need to move beyond acute responses to test the influence of a locomotor exercise program alone (i.e., without an associated stimulation protocol) on the plasticity of brain neural networks. Fourth, investigations should focus on populations specificities (i.e., pathologies and age) both in relation to the most appropriate type(s) of exercise for each population and to the mechanisms underlying neuroplasticity-mediated functional outcomes. Fifth and final, one way of testing the endogenous hypothesis behind the neuroplastic effects of eccentric exercise is to try to fathom the influence of muscle afferent feedback. Solutions may include the use of paired-pulse TMS following locomotor eccentric exercise performed under pharmacological blockade of type III/IV afferences, or during mental imagery of eccentric exercise. Either downhill walking/running or eccentric cycling can be used to carry out these perspectives, yet eccentric cycling seems more suited to the use of TMS during exercise provided the relative stability of the participant's head it allows.

Conclusion
Conventional and eccentric locomotor exercises can both lead to decreases in intracortical inhibition and increases in intracortical facilitation, which is also the case of the learning of a basic motor task. The changes induced by conventional exercise seem to originate mainly from hemodynamic mechanisms causing the release of neurotrophic factors, while those triggered by locomotor eccentric exercise seem to be the result of repeated activation of neural networks, and maybe of hemodynamic processes as well. Furthermore, the low cardiorespiratory response to eccentric contractions adds to the relevance of this exercise modality as an alternative to conventional rehabilitation protocols in weak patients.
In short, we believe it is time for clinical science to widen its focus to scrutinize eccentric exercise-induced neuroplasticity.
Author contributions All the authors decided of the review boundaries. PC and YG drafted the manuscript. YG and RL drew the figures. PC finalized the manuscript. All the authors critically revised and approved the final version of the manuscript.

Conflict of interest
The authors declare no conflict of interest.