Chronic exercise changes the baseline properties of central monoaminergic systems, identified neurons and related behaviors

Chronic exercise changes the baseline properties of central monoaminergic systems, identified neurons and related behaviors | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Chronic exercise changes the baseline properties of central monoaminergic systems, identified neurons and related behaviors Andrey Sorminskiy, Aisha Atnagulova, Dmitry Vorontsov, Victoria Melnikova, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9290981/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Physical exercise influences monoaminergic and neurotrophic systems in the brains of mammals and some invertebrates. Little is known about how a specific neuron adapts its biophysical properties to acute or repeated physical activity. Here we tested the hypothesis that central monoaminergic neurons develop a state of readiness for exercise by changing their baseline biophysical properties during regular training. In the mollusc Lymnaea stagnalis, we compared the activity of central monoaminergic neurons and brain monoamine levels immediately after a single two-hour exercise (crawling in low water) and one day after completion of the two-week daily two-hour exercise. Under both acute and baseline chronic conditions, serotonergic neurons that control locomotion were depolarized both in situ and being completely isolated. The dopaminergic neuron that controls respiration was hyperpolarized, and dopamine levels in the brain were reduced under both acute and chronic conditions, while a two-hour rest after acute exercise abolished these effects. Monoamine-dependent behaviors were also altered by regular exercise: the speed of locomotion, consumption and oviposition increased, while growth rate decreased. Our findings suggest that regular exercise alters the baseline biophysical properties of central monoaminergic neurons, potentially through an anticipatory mechanism preparing the nervous system for subsequent exercise. Biological sciences/Neuroscience Biological sciences/Physiology serotonin dopamine single neurons exercise locomotion adaptation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Regular physical exercise exerts a multifaceted effect on behavior and the body. Changes in the musculoskeletal system have been well studied. These changes are known to be determined by the type of exercise: short-term strength training leads to greater increases in muscle mass and strength, while longer-term aerobic exercise improves endurance, allowing adaptation to the training stimulus. Adaptation manifests itself in structural and metabolic changes of muscle fibers: short-term exercise can be provided by the energy derived from rapid anaerobic glycolytic breakdown of glucose, while prolonged exercise requires a slower but more efficient process of oxidative phosphorylation occurring in the mitochondria [ 1 ]. At the same time, it is generally understood that changes in the muscular system are only the tip of the iceberg, while the main events that determine the impact of exercise on behavior occur in the brain [ 2 , 3 , 4 ]. Less is known about neuronal events accompanying exercise, especially with regard to individually identified neurons. Although it is known that exercise activates neurogenesis and influences neurotransmitter, neurotrophic, and neuromodulatory systems [ 5 , 6 ], as well as gene expression in the mammalian brain [ 7 ], it remains unclear how a specific neuron adapts its properties to acute or repeated physical activity. Recent work in mammals has begun to address this issue by demonstrating that repeated exercise enhances synaptic input strength and increases the intrinsic excitability of defined hypothalamic neurons (VMH SF1), and that the activity of these neurons is causally required for endurance adaptations [ 8 ]. These findings indicate that exercise-induced plasticity at the level of identified neuronal populations can directly mediate physiological adaptation, but the cellular mechanisms remain difficult to dissect in vertebrate systems. The relationship between presumed changes in specific neurons and subsequent changes in other behaviors, such as learning, decision-making, feeding, and reproduction, that are known to be influenced by acute and chronic exercise [ 9 , 10 , 11 ], remains to be elucidated. These questions are challenging to address in the complex mammalian brain. However, several simpler models have recently emerged for studying changes in the nervous system during exercise [ 12 ]. We previously found that, in the mollusc L. stagnalis , a single two-hour experience of intense muscular crawling in low water facilitates proactive behaviors, decision-making in a novel environment [ 13 , 14 ] and enhances reproduction [ 15 ]. Such exercise affected the expression of a number of genes in the snail nervous system [ 16 ]. Notably, dozens of homologous genes were also affected by exercise in the mouse brain [ 7 , 16 ]. By taking advantage of the Lymnaea model for cellular analysis, we previously reported the accompanying changes in the membrane potential and the activity of identified central serotonergic neurons involved in locomotion [ 17 ]. These changes persisted even after the complete isolation of the neurons from the central ganglia. In addition, we obtained evidence for changes in the dopaminergic system following exercise (17). In Lymnaea , the involvement of dopaminergic mechanisms in exercise is plausible since the giant pedal dopaminergic neuron RPeD1 is a component of the respiratory central pattern generator (CPG) in L. stagnalis , while an increased respiratory rate accompanies exercise in most animals [ 18 , 19 , 20 , 21 , 22 ]. The aim of this study was to test the hypothesis that monoaminergic neurons develop a state of readiness for exercise during chronic intense locomotion by changing their baseline properties. To test this, we used the same Lymnaea model and compared the activity of identified monoaminergic neurons and brain monoamine levels immediately after a single acute two-hour exercise and one day after the end of the two-week daily two-hour exercise protocol. To analyze the neuronal effects of exercise, we selected serotonergic neurons of the PeA cluster that directly control locomotion [ 22 ] and the unpaired dopaminergic neuron RPeD1, which is involved in the generation of the respiratory rhythm [ 18 ]. Additionally, we recorded and analyzed changes in the locomotor, feeding, and reproductive behaviors during chronic regular exercise. Results Effects of single acute exercise Exercise enhances breathing in L. stagnalis An increased breathing rate accompanies intense locomotion in most animals studied in this context [ 27 , 28 , 29 ]. We tested whether the same occurs during intense crawling in L. stagnalis . The mean and the total duration of pneumostome openings (PNO, Fig. 1 A), summed over 10 min, were significantly higher during the last 10 minutes compared to the first 10 minutes of the 2-h locomotor session (Fig. 1 C, D, n = 20, Wilcoxon test: p = 0.02, 0.027, correspondingly). The number of PNOs was significantly higher at the end of the exercise (Fig. 1 E; n = 20; paired Wilcoxon signed-rank test: p = 0.01). Thus, breathing rate increased during 2 hours of intense locomotion. In the control group (n = 20, no differences were detected in PNO frequency or duration between the beginning and end of the same 2-h interval (Wilcoxon test for differences in opening frequency in the control: p = 0.328; for total duration of open state of pneumostome: p = 0.1; and for average duration: p = 0.13). This is consistent with the increased locomotion speed observed after two hours of low-water crawling in L. stagnalis [ 13 ] and with the previously reported coordination of respiratory and locomotor rhythms [ 27 ]. Acute exercise decreases dopamine content in the pedal ganglia The dopamine (DA) content in the pedal ganglia decreased significantly immediately after the acute 2-h exercise (t-test, p = 0.001, Fig. 1 F, the left group of bars). In contrast, DA content in the cerebral ganglia did not differ between control and exercised snails (Mann–Whitney U Statistic = 15; p = 0.699, Fig. 1 F, the right group of bars). Two hours of rest in deep water after the acute exercise abolished the decrease of DA in pedal ganglia. DA content was 82.9 ± 6.5 pmol in exercised-then-rested snails (n = 6) and 67.6 ± 5.2 pmol in control snails (n = 6) kept for four hours in deep water and handled twice to match the exercised-then-rested group. A non-significant trend toward a rebound increase of DA content was observed in exercised-then-rested snails (p = 0.093). Thus, the exercise-induced changes in DA content were transient and were not maintained after a single exercise bout. Acute exercise decreases the tonic activity of the dopaminergic RPeD1 neuron RPeD1 neuron (Fig. 2 A, B) is known to have several modes of activity. It can generate spikes tonically in the absence of respiratory rhythm. It is thought that sensory cells in the pneumostome-osphradium region, activated by chemosensory stimuli (e.g., hypoxia), provide strong excitatory afferent input to RPeD1 [ 18 ]. Through synaptic connections within the respiratory CPG, activated RPeD1 initiates CPG activity underlying the respiratory rhythm. During respiratory CPG activity, RPeD1 becomes hyperpolarized, ceases tonic firing, and exhibits rhythmic bursting driven by EPSP bursts from respiratory CPG interneurons [ 18 , 27 ]. After intense locomotion, RPeD1 neurons (n = 18) were in a more hyperpolarized state (Mann-Whitney U-test: p = 0.013, Fig. 3 A) and exhibited significantly lower spike frequency (Mann-Whitney U-test: p = 0.007, Fig. 3 B,С) than neurons from control snails (n = 18) recorded in parallel. The hyperpolarizing effect of acute exercise on RPeD1 cannot be explained by reduced dopamine content of in the pedal ganglia The giant dopaminergic RPeD1 neuron expresses dopamine receptors [ 25 ]. Given this finding, reduced DA content in the pedal ganglia of exercised snails could influence the spiking activity and membrane potential of RPeD1. To test this, we examined the effect of exogenous DA on RPeD1 neurons in the CNS isolated from control and exercised snails. In both control (n = 8) and exercised (n = 8) snails, DA hyperpolarized RPeD1 neurons (Fig. 3С-F). After DA application, the difference between preparations from control and exercised snails became even more evident: DA completely suppressed tonic firing of RPeD1 from exercised snails, whereas tonic activity persisted in control preparations (Fig. 3 D). This difference might, in part, reflect pre-existing baseline differences between the groups. Nevertheless, the possibility of an elevated sensitivity of RPeD1 to DA (and potentially enhanced autoinhibitory feedback via DA receptors) in preparations from exercised snails cannot be dismissed. Taken together, these findings suggest that the post-exercise hyperpolarization of RPeD1 is not a consequence of reduced DA concentrations within the pedal ganglia. Rather, it may reflect training-induced alterations in intrinsic membrane properties that set the resting membrane potential and/or an increased sensitivity to dopamine’s hyperpolarizing action. Effects of chronic exercise Two-week regular exercise decreased baseline DA content in the pedal ganglia We measured dopamine content 24 h after the final session of the two-week daily exercise protocol. Baseline DA levels in the pedal ganglia were significantly reduced compared with controls (n = 12 per group, p = 0.039; Fig. 4 A, right panel). In contrast, levels of the DA precursor L-3,4-dihydroxyphenylalanine (L-DOPA) did not differ between groups (Fig. 4 B). This pattern is consistent with increased DA turnover rather than reduced synthesis, although synthesis and degradation were not directly measured. No significant changes were detected in the cerebral ganglia for either DA or L-DOPA. Thus, chronic exercise affected pedal ganglia DA in the same direction as acute exercise. Chronic exercise for two weeks decreased baseline activity of dopaminergic RPeD1 neuron After the two-week exercise protocol, RPeD1 was hyperpolarized (n = 11 per group, p = 0.007; Fig. 5 A), and its firing activity was significantly reduced (n = 11 per group, p = 0.002; Fig. 5 B, C); in most preparations, the neuron was silent. This state resembled the response observed after a single 2-h acute exercise bout. Chronic exercise for two weeks did not alter baseline serotonin levels The effect of a single acute exercise bout on serotonin (5-HT) and its catabolites in the pedal and cerebral ganglia have been described previously [ 14 ]. Both ganglia contain numerous serotonergic neurons, including those involved in locomotion. Acute exercise did not significantly change 5-HT levels; however, during the post-exercise rest period, levels of all measured serotonin catabolites were significantly increased [ 14 ]. Here we examined whether chronic exercise affects serotonin levels. No significant changes were detected in either the cerebral ganglia or the pedal ganglia after the two-week exercise protocol (Fig. 4 C). In the pedal ganglia, there was a trend toward decreased 5-HT levels (p = 0.06). Chronic exercise for two weeks induced depolarization of serotonergic PeA cluster neurons in situ and after complete isolation The effect of single acute exercise on electrical properties of serotonergic PeA-cluster neurons in situ (within the CNS) and after complete isolation have been described earlier [ 13 , 17 ]. Depolarization and higher spiking activity were observed in these cells after acute exercise, which agreed with behaviorally observed increase in the locomotion speed [ 13 , 17 ]. After chronic exercise, the serotonergic neurons of PeA cluster were depolarized (-48 ± 1.98 mV versus − 54.7 ± 3.3 mV for control; n = 38, 33; t = 3.34, p = 0.0013, Fig. 6 A), but their spiking activity remained virtually unchanged (40.2 ± 9.4 AP/min versus 30.9 ± 6.4 AP/min for control; t = 1.6; p = 0.1, Fig. 6 B). After complete isolation of the PeA cluster neurons from the CNS (Fig. 6 C, D), group differences became more pronounced. In chronically exercised snails, isolated serotonergic neurons demonstrated significant depolarization (-46.6 ± 4.4 mV versus − 55.3 ± 5.8 for control; n = 18, 19; Mann-Whitney U-test z = 2.3, p = 0.019, (normality test p < 0.05), Fig. 6 E) and higher spiking rate (34.9 ± 9.6 AP/min versus 16.7 ± 9.5 AP/min for control; Mann-Whitney U-test z = 2.7, p = 0.007, (normality test p < 0.05), Fig. 6 F). These effects resemble those previously reported after a single acute exercise bout [ 17 ]. Behavioral changes produced by two-week chronic exercise Locomotion speed was analyzed separately in each of the four experimental groups (Fig. 7 A, C, D). For statistical analysis (Fig. 7 B), data from four groups were pooled. Mean locomotion speed was significantly increased after one week of exercise compared with the first day (Fig. 7 B). On the final day of two-week protocol, the increase in speed was smaller than after one week but remained statistically significant (Fig. 7 B). The within-session dynamics of locomotion speed during the 2-h training period also differed on the final day compared with the first day (Fig. 7 D compare to Fig. 7 C). On the first day, locomotion started at a lower speed and increased toward the end of the session. In contrast, on the final day animals exhibited a significantly higher initial speed, which progressively decreased over the session. Locomotion in a completely dry arena We also tested snail behavior in a completely dry, asymmetrically lit arena [ 13 , 14 ] one day after the end of the two-week exercise protocol, i.e., under the same conditions as those used for electrophysiological and HPLC experiments. Trained snails exhibited higher speeds than control snails, as shown in the Fig. 7 E ((n = 12 per group) Wilcoxon paired test for means at each time interval, p = 0.005). This result is consistent with the increased locomotion speed observed during training in low water. However, no difference was found in any other behavioral parameters between control and experimental snails, including the number of orienting turns (p = 0.4, t-test, normal distribution) or time to the border of the arena (p = 0.33, U-test). The dispersion of some parameters was lower in the exercised group, such as the time spent in the central zone (Fligner p value for equal dispersion 0.005). Weight and growth Significant differences in body mass were detected after the two-week chronic exercise protocol. Exercised snails weighed less than control (1.67 ± 0.14 g versus 2.02 ± 0.13 g in the control group (t = 4; p = 0.0003, (normality test p > 0.1), Fig. 8 A). Possible impact of weight difference on locomotion speed in experimental and control animals To test the possible influence of weight differences on the speed of locomotion in experimental and control snails, we compared the behavior of intact large (5.2 ± 0.5g) and small (1.7 ± 0.3g) animals in the same dry arena. There was no significant difference between small and large snails in any of the evaluated parameters: distance, velocity, time spent in the center, first exit, number of turns (p > 0.09). The larger snails showed a trend for a higher speed of locomotion (0,054 cm/s versus 0,045 cm/s; p = 0.0952), which is opposite to the difference observed between control and TWRE snails. Thus, we conclude that the difference in body weight does not explain the higher speed of locomotion observed in chronically exercised snails. Consumption To determine whether reduced growth could be explained by lower food intake, consumption was measured in an additional experimental series. Contrary to this hypothesis, food intake was higher in the exercised group throughout the 13-days observation period. The mean mass of consumed food was 3.13 g in control versus 4.7 g in exercised snails (n = 13; Z = 2.9, p = 0.0037 paired Wilcoxon test, Fig. 8 B). Oviposition Clean water is known to stimulate egg laying in L. stagnalis [ 15 , 30 , 31 ]. This effect was evident throughout the entire chronic exercise experiment, when the water was replaced in containers of both control and exercised groups (see Fig. 8 C, days 1, 4, 8, 11, 14). The number of egg clutches was higher but only as tendency (t-test, p = 0.05) in the exercised group (Fig. 9D). Average number of eggs over two weeks was 746.5 ± 80 in control and 1006.5 ± 55 in exercised snails. The number of eggs per clutch did not differ significantly (t-test, p = 0.09). Discussion The primary aim of this study was to test the hypothesis that monoaminergic neurons develop a state of readiness for exercise during regular intense locomotion by altering their baseline properties. Towards this aim, we compare the activity of identified neurons involved in locomotion and respiration, as well as monoamine levels, immediately after a single 2-h exercise session and 24 h after completing a two-week daily exercise protocol. We observe similar changes in monoaminergic neuron state and ganglionic monoamine content immediately after a single exercise bout and 24 h after two weeks of daily exercise. In both cases, the serotonergic neurons of PeA cluster, which are related to locomotion, were depolarized, both in situ and after their complete isolation, while no significant changes were observed in the serotonin content. At the same time, the respiration-related dopaminergic neuron RPeD1 was hyperpolarized and dopamine content was decreased in both cases. These findings strongly support the idea that baseline properties of monoaminergic systems are affected by regular exercise. The similarity of these changes with the effects of acute exercise may suggest an anticipatory/predictive mechanism. Notably, post-training measurements were performed at the time of day when the next exercise session would normally occur. We argue against a single-switch explanation in which the first exercise bout produces a persistent state change. Consistent with this, a 2-h rest period after a single acute exercise bout produces changes opposite to those observed immediately after exercise [ 14 , 16 , 17 ]. The great pond snail L. stagnalis is a relatively new model for studying the effects of exercise on the brain. Importantly, this model incorporates an ethologically and ecologically relevant behavioral paradigm. The form of intense locomotion, which is treated here as exercise, is specific for this snail. It is activated when the snail is in shallow water or actively crawls onto the land. L.stagnalis has already been shown to share many similarities with mammals in the behavioral effects of exercise, such as reduced fearfulness, increased activity and determination in a new environment, facilitated decision-making [ 13 , 14 ]. Similarities with mammals also include the involvement of monoaminergic systems in these effects [ 5 , 14 , 17 ] and are present even at the level of gene expression: in our recent study [ 16 ], we discovered a non-random overlap of gene subsets whose brain expression is influenced by exercise both in mice [ 7 ] and snails. A number of similar functional clusters of genes responded to exercise in these distinctly-related species [ 16 ]. One of the main advantages of L. stagnalis as a model animal is that it allows tracing the entire chain of effects associated with exercise in the central nervous system, starting from the very first stage – changes in the properties of central serotonergic neurons of the PeA cluster, which send projections to the sole and to the pedal cilia and thus directly regulate locomotion [ 22 ]. So far, the shift in the individual biophysical characteristics of identified neurons caused by exercise has been directly demonstrated only in Lymnaea [ 15 , 17 ]. In this study, changes in the membrane potential are detected in serotonergic neurons of the PeA cluster and in the dopaminergic neuron RPeD1 both immediately after a single acute exercise and after two weeks of chronic exercise. In this regard, it should be noted that the expression level of the UNC80 gene, which plays a role in regulating membrane potential, was affected by exercise in both mice and snails [ 16 ]. Unc80 is an evolutionarily conserved gene. It encodes a protein that is a component of the voltage-independent leakage ion channel complex, which plays an important role in establishing resting membrane potential in neurons of both invertebrates and mammals [ 32 ]. It is widely expressed in the nervous system of the nematode C. elegans , and worms with the knockout of the Unc80 gene are capable of crawling but are unable to switch to more intense and rapid locomotion, such as swimming [ 32 ]. In humans, mutations in Unc80 are associated with congenital infantile encephalopathy, mental retardation, and growth problems [ 33 ]. We hypothesize that the membrane potential of some key neurons related to locomotion is affected by exercise not only in the mollusk, where we directly demonstrate this, but also in the mammalian brain, where it still remains challenging to track changes in the biophysical properties of individual neurons. This is consistent with the exercise-induced Unc80 expression changes reported in the mammalian brain. Our assumption is supported by recent evidence showing that repeated exercise increases the excitability of VMH SF1 neurons in mice, and that suppression of their post-exercise activity prevents the development of endurance adaptations [ 8 ]. Thus, exercise-induced shifts in baseline neuronal excitability appear to represent a conserved mechanism contributing to systemic effects of training. The possible role of 5-HT in the modulating effects of exercise has been investigated and discussed earlier [ 14 ]. Briefly, an increased release of 5-HT can explain only some of the known effects of intense locomotion in L. stagnalis . Serotonin increases the speed of locomotion in novel environment and can contribute to the higher oviposition rate observed after a single exercise [ 15 , 34 ]. However, elevated 5-HT levels do not explain changes in goal-oriented behavior and decision-making [ 14 ]. In this study, we initiated a cellular analysis of the potential involvement of respiratory system neurons in mediating the effects of exercise. It is generally known that intense physical activity has a significant impact on the respiratory system because of the close relationship between body movements, oxygen demand and breathing. One of the main mechanisms of this interaction is the coordination of breathing rhythms with walking or running. For example, recent research has shown that a specialized region of the brain called the mesencephalic locomotor area can activate respiratory centers in advance, before the blood carbon dioxide levels rise [ 29 ]. That discovery suggests that an organism increases breathing ahead of exercise, preparing itself for increased physical exertion, and that central neural control of respiration demonstrates significant autonomy from peripheral chemoreceptors [ 29 ]. Of importance is the locomotor-respiratory coordination. In humans, breathing is frequently synchronized with the stepping rhythm, reducing overall energy expenditure [ 28 ]. Such coordination has also been demonstrated in L. stagnalis , which possesses neurons that simultaneously regulate respiration and intense locomotion [ 18 , 27 ]. One such neuron investigated here is RPeD1 [ 18 ]. Immunohistochemical studies have shown that this neuron contains tyrosine hydroxylase (Fig. 2 ), confirming its dopaminergic nature [ 35 , 36 ]. RPeD1 projects into the right and left pedal ganglia and also sends axons to the cerebral and pleural ganglia via connecting nerves, forming synaptic connections with other neurons involved in the regulation of respiration and locomotion [ 18 , 36 ]. Neurons displaying D1 receptor-like immunoreactivity have been observed in all ganglia of L. stagnalis but particularly in the pedal ones [ 37 ]. It should be noted that, functionally, the Lymnaea pedal dopaminergic system is involved not only in the regulation of respiratory rhythm [ 37 , 38 ]. Thus, studies have shown that dopaminergic neurons, including RPeD1, play a key role in the formation of long-term memory, particularly in the context of operant breathing learning [ 39 ]. Dopaminergic neurons are also present in various peripheral tissues [ 40 ], peripheral sensory dopaminergic axons innervate the pedal ganglia in molluscs [ 41 ]. In the pedal cilia of L.stagnalis , dopamine exerts the effect opposite to serotonin, namely, inhibits cilia beating and decreases the speed of locomotion [ 42 , 43 ]. Here we show that respiratory rhythm indeed increases toward the end of a two-hour exercise in the L.stagnalis . Surprisingly, the RPeD1 neuron, which is believed to initiate and maintain the respiratory rhythm generation, was hyperpolarized in trained snails. As was mentioned above, this neuron exhibits diverse activity patterns. In the absence of a respiratory rhythm, it can fire action potentials continuously. It is hypothesized that sensory cells located in the pneumostome-osphradium region, when stimulated by chemosensory cues such as hypoxia, deliver robust excitatory afferent input to the RPeD1 neuron [ 18 ]. This activation of RPeD1 is thought to trigger the CPG activity that drives the respiratory rhythm. Nevertheless, during the active generation of the respiratory CPG, the RPeD1 neuron itself undergoes hyperpolarization. This hyperpolarization suppresses its intrinsic tonic firing, causing it to display rhythmic bursting activity instead, which is driven by incoming bursts of excitatory postsynaptic potentials (EPSPs) originating from the respiratory CPG interneurons [ 18 , 27 ]. The same hyperpolarizing effect is produced by dopamine as we show here and also earlier [ 25 ]. In the latter study, the inhibiting effect of dopamine on autoreceptors of RPeD1 is confirmed in completely isolated RPeD1 neurons. Thus, the hyperpolarized state observed in trained snails could reflect the state of RPeD1 during respiratory rhythm generation. However, this effect cannot be explained by reduced dopamine levels in the pedal ganglia after exercise, because dopamine itself hyperpolarizes RPeD1. Instead, it appears to be a consequence of alterations in intrinsic mechanisms governing membrane potential, and/or a substantially elevated responsiveness to dopamine's hyperpolarizing influence. Notably, the same combination of effects (decreased dopamine content and hyperpolarized membrane potential of RPeD1) was observed after two weeks of regular exercise, but after 24 hours since the last exercise episode. Chronic exercise not only altered the baseline properties of RPeD1 but also made them resemble the acute exercise state even 24 h after the last session. This finding agrees with the idea that “organism increases breathing in advance, preparing itself for increased physical exertion” [ 29 ]. Interestingly, this neuronal “preparedness” is consistent with the increased locomotion speed observed in chronically exercised snails. By the end of the second week, locomotion speed remained elevated relative to day 1. The overall speed profile changed over the two hours of exercise: while, on the first day, the animals started at comparatively low speed and finished at higher speed, on the last day of chronic exercise, the opposite pattern was observed: a high start and a low finish. It is possible that the exercise protocol that we chose was somewhat excessive, which was reflected in a reduced average speed after 14 days compared to seven days of exercise, as well as in the progressive slowing over two hours of exercise observed on the last day. The behavior of chronically exercised and control snails in novel, completely dry conditions confirmed the ability of trained snails to develop faster locomotion even under unfamiliar stressful conditions. This effect occurred even though they had a lower body mass than controls. Monoaminergic systems are known to affect other behaviors in L. stagnalis , such as feeding and reproduction [ 34 , 44 ]. In addition, we propose that there may be other parameters beyond the monoamine levels and the activity of monoaminergic neurons that undergo changes during the exercise. To address this, during the two-week chronic exercise, in addition to locomotion we analyzed several other behaviors and states of the snails. We found that exercised snails exhibited higher food consumption and laid more eggs. However, their growth apparently slowed compared to the control group. Increased locomotor, feeding, and reproductive activities are consistent with the known effects of metabolic intensification induced by intense locomotion in other organisms, vertebrates [ 9 , 10 , 11 ] and invertebrates [ 12 , 45 ]. Interestingly, in L. stagnalis these effects were observed also after single exercise session [ 13 , 15 ]. Meanwhile, the significant inhibition of growth that compensated for these activities suggests the possible involvement of the brain insulin signaling in this shift of organismal state. Indeed, in many animals, neuronally-produced insulin determines the balance between energy expenditure, reproduction, and growth [ 46 ]. In L. stagnalis , removal of specific brain regions, lateral lobes, containing insulin neurons results in the decrease in the mollusk's size [ 47 ] and activates reproduction [ 48 ]. The suppression of body growth appears to have been connected to the boosting of female reproductive output. Given that the consistent generation of egg masses, intense locomotion and the expansion of body size necessitate significant resource and energy expenditure, it's probable that some of these functions are mutually exclusive. Changes in several behaviors besides locomotion that we observed after exercise suggest that other neurons, probably related to feeding and reproduction, as well as other neuromodulatory systems, such as the insulin system, can similarly adjust their properties to the training. In conclusion, we present a new invertebrate model for studying the effects and mechanisms of chronic exercise. We show that behavioral effects of regular training in L. stagnalis can be traced to changes in the properties of identified central neurons. Our results support the idea that regular exercise induces an anticipatory adjustment of baseline properties in monoaminergic neurons. Materials and Methods Animals Mature specimens of Lymnaea stagnalis were taken from a breeding colony kept in dechlorinated tap water at room temperature and regularly fed with lettuce ad libitum. Three-month-old snails weighing 1.7 ± 0.2 g were used for the experiments. Behavioral experiments Acute exercise Experimental snails were placed for two hours in flat white trays, 25 x 50 cm, filled with 1 cm-deep water, taken from the colony aquarium as in [ 12 , 13 , 14 ]. Under these conditions, terrestrial locomotion (crawling) is induced, which is energetically more demanding than underwater ciliary locomotion. In parallel, control snails were removed from the domestic aquarium and placed into a new container filled with water from the colony aquarium where they could drift normally. After two hours, experimental and control snails were dissected under MgCl 2 0.1 M anesthesia for electrophysiology or HPLC analysis of monoamine content. In an additional control series, the experimental animals were allowed to rest in deep water for two hours after exercise, and then were dissected for HPLC analysis. Analysis of respiratory behavior during two hours exercise The number and duration of pneumostome openings (PNO, Fig. 1 A) were recorded during the first and last 10 minutes of the two-hour locomotor activity. A video camera positioned beneath the transparent floor of the arena allowed continuous recording of PNOs during locomotion (Fig. 1 B). For each snail, the frequency of PNOs was calculated from the video recordings based on these time intervals. Chronic exercise Prior to the experiment, we prepared eight groups of snails (n = 8 per group) of the same size (10 weeks old). Animals were taken from their home aquarium and placed into 2.5 L containers (one group per container) filled with clean settled water and fed with lettuce ad libitum. During two days of adaptation to these novel conditions, we evaluated oviposition in all groups (the number of clutches and individual eggs) to exclude initial differences in these parameters between the groups. The groups were then assigned to either the experimental or control protocol. Four experimental groups (eight snails per group) were placed in flat white trays (25 × 50 cm) filled with 1 cm-deep water (one group per tray). After 2 hours of crawling in low water, the experimental groups were returned to their containers until the next day. In parallel, four groups of control snails were handled and returned to their containers. The same procedure was repeated daily at the same time (12:00) for two weeks. Every fourth day, the water in all containers was replaced with fresh water. Crawling snails were video-recorded from above at 2 frames per second. The resulting videos were pre-processed in VirtualDub and then automatically tracked in FIJI [ 23 ] using the TrackMate plugin [ 24 ]. Behavioral test on the dry surface One day after the end of the two-week exercise protocol, we compared the behavior of trained (two-week regular exercise, TWRE) and control snails under new conditions, in a completely dry arena as in [ 13 ]. This experiment was conducted to test the hypothesis that regular exercise under low water conditions pre-adapts snails to completely dry conditions. Individual snails (one at a time) were placed into a rectangular arena (60 x 45 cm) on a flat, dry plastic surface that aimed to simulate the potentially desiccating condition of being out of water (as in 13, Fig. 1 B). In such a situation, a snail is expected to select a direction of movement as quickly as possible to return to an aquatic environment and to avoid desiccation. One of the shorter walls of the arena was made of translucent plastic to provide the asymmetric illumination. All other walls were made of black opaque plastic. A white LED lamp was placed to the translucent side of the arena as a source of light. The light intensity at the arena’s center (measured 3 cm above the surface with the Proskit MT-4017 luxmeter) was made equal to 80 lux, and it was 120 and 20 lux near the light and the dark walls, respectively. No measurable amount of light came to the arena from below. The movements of each snail were recorded at 15 frames per second for 15 minutes with a video camera placed below the transparent plastic bottom of the arena. The recordings were tracked using the EthoVision XT software (Noldus, the Netherlands) and independently scored manually with RealTimer (Openscience, Russia). Snail turns were counted manually. The traces left by the crawling snails were removed with a clean paper towel before each new test. During the analysis, a centered circle zone (10 x 10 cm) that limited the track and scoring analysis was defined. This zone was used to exclude from the analysis a snail's movements near the physical borders of the arena, especially near the brightly lit wall. We evaluated (i) mean locomotion velocity over 1-min intervals (ii) the number of rotations performed by the snail (iii) the time taken to crawl to the virtual border of the arena as in [ 14 ]. When a snail did not reach the virtual border, the time was given the value of the total time of observation, which was 15 minutes. Manual scoring was performed blind to group assignment. Analysis of oviposition 24 hours following each exercise session, the number of egg clutches, the number of eggs per clutch and the total number of eggs laid in the exercised and control groups were counted. Analysis of consumption In an additional experiment with two cohorts of snails (n = 16 for control and n = 16 for experiment) we controlled feeding behavior during the two-week daily exercise. The procedure of exercise was the same as described above. Each day, immediately after the exercise session, each experimental and control group of snails received 5 g of fresh lettuce. After 12 hours, the remaining lettuce was removed from the containers and weighed. The consumed mass was calculated. Weight gain analysis Both the control and exercised snails were weighed after the two-week chronic exercise. The distribution was tested for normality, and then the significance of differences between groups was evaluated by t-test. Electrophysiology and cell isolation In each experiment, two snails (experimental and control) were anesthetized by injection of 0.1 mM MgCl 2 , followed by dissection of the central ganglia (central nervous system, CNS). Both CNS preparations were placed into a 3 mg/ml solution of pronase E (Sigma) for 15 min, washed in a standard snail Ringer’s solution (50 mM NaCl, 1.6 mM KCl, 4 mM CaCl 2 , 8 mM MgCl 2 , 10 mM Tris, pH 7.6) and then both CNS were pinned in a 40 mm Sylgard-bottomed chamber at a distance of approximately 10 mm between them. The connective tissue sheath was then removed from the pedal ganglia in both CNS. Visual identification of the RPeD1 and PeA neurons was performed based on their location, size and coloration (Fig. 2 A, B). The neuron selected for recording was impaled with a standard glass microelectrode (10–20 MΩ) filled with 3 M KCl. A standard setup for microelectrode recording was used. The electrophysiological recordings were stored in computer files using a home-made software. Mechanical isolation of a neuron was performed according to the previously developed method [ 25 ]. Using the intracellular microelectrode as a handle, the neuron was gently pulled out of the ganglion tissue until separation of the proximal neurite from the neuropile was achieved. The electrical activity of the cell was continuously monitored during isolation. The cells, which demonstrated membrane injury, were excluded from the following experiments. High Performance Liquid Chromatography Central ganglia were quickly dissected on ice, homogenized using an ultrasonic homogenizer (Bandelin Sonopuls, Burladingen, Germany) at 4°C in 0.1 M HClO₄, and centrifuged at 10,000 × g for 20 min at 4°C. The supernatant was collected and stored at − 80°C prior to monoamine quantification. An Agilent 1260 Infinity II HPLC system (Agilent Technologies Inc., Waldbronn, Germany), equipped with a fluorescence detector (FLD), was used for monoamine analysis. Analytes were separated using a reverse-phase InfinityLab Poroshell 120 EC-C18 column (100 mm × 4.6 mm, 2.7 µm particle size; Agilent Technologies Inc., Germany). The column was thermostated at 30°C. The mobile phase consisted of 0.1 M citrate–phosphate buffer, 0.25 mM sodium 1-octanesulfonate, 0.1 M EDTA, and 7% acetonitrile (pH = 2.56) (all reagents purchased from Sigma-Aldrich, St. Louis, MO, USA). The mobile phase flow rate was 1 mL/min. FLD detection was carried out at an excitation wavelength of 285 nm, and emission was recorded at 310 nm. Peaks were identified based on retention times relative to standard solutions, and monoamine concentrations were calculated from the ratio of peak areas relative to the standards. In some experiments (see Fig. 3 ), DA, DOPA, and 5-HT were quantified using an electrochemical detector (Decade Elite, Antec, the Netherlands) equipped with a glassy carbon flow cell and a salt-bridge Ag/AgCl reference electrode, with the potential set at + 0.85 V. Monoamine content is presented as pmol per ganglion, as the ganglia are very small and weighing the samples would increase measurement error. Data analysis Normality of the data was assessed prior to statistical testing. Depending on the distribution, either the Mann–Whitney U test or the Student’s t-test was used to evaluate differences between independent groups. For dependent samples, the paired Wilcoxon signed-rank test was applied. Statistical analysis and figure preparation were performed using PAST [ 26 ] or the R Project for Statistical Computing. For the HPLC data, statistical analysis was performed using GraphPad Prism version 8.1.1. All values are presented as mean ± SEM. Statistical significance was set at p < 0.05. Declarations Author Contributions: Conceptualization, V.D. and D.V.; investigation, A.S., A.A., I.Ch., V.M., Yu.N., An. A., I.Z. and M.M.; writing—original draft preparation, V.D.; writing—review and editing, D.V., A.A. and A.S.; visualization, V.D., D.V. and A.S.; project administration, V.D. and I.S. All authors have read and agreed to the published version of the manuscript. Funding: Supported by RSF 25-14-00147. This research was conducted using the equipment of the Core Centrum of the Institute of Developmental Biology RAS. Institutional Review Board Statement: Not applicable. Data Availability Statement: The data that support the findings of this study are available within the article. Further information can be obtained from the corresponding author upon reasonable request. Conflicts of Interest: The authors declare no conflicts of interest. References Smith, J. A. B., Murach, K. A., Dyar, K. A. & Zierath, J. R. 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Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 12 May, 2026 Reviews received at journal 03 May, 2026 Reviews received at journal 27 Apr, 2026 Reviewers agreed at journal 26 Apr, 2026 Reviewers agreed at journal 23 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviewers invited by journal 20 Apr, 2026 Editor assigned by journal 16 Apr, 2026 Submission checks completed at journal 06 Apr, 2026 First submitted to journal 06 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9290981","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":630499777,"identity":"968346ee-e9f9-4e92-bc7c-4754d80f591e","order_by":0,"name":"Andrey Sorminskiy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYHACA4YEHhs5xvYGENuCSC0PZNKMmXsOgNgSxGlhfGBzOJF9RgKIQ4QW/tnN2yQScpgTeGc+v7rhR4EEA397dwJeLRJ3jpVJJJxhy5OcnVN2swfoMIkzZzfgt+ZGjplEYg9PseHsnLQbPEAtBhK5+LXIg7X8k0jcf/NM2s0/xGgxAGlJ4DFIbJzBfuw2UbYY3kgrtkjgSTBm7Mlhuy1jIMFD0C9yN5I33vzB8x8Ylcef3Xzzx0aOv72XgPcZGFigccFjACYJKQcB5g8Qmv0BMapHwSgYBaNgBAIAU7FKeC23VMYAAAAASUVORK5CYII=","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":true,"prefix":"","firstName":"Andrey","middleName":"","lastName":"Sorminskiy","suffix":""},{"id":630499780,"identity":"315aa501-7b8f-4c10-b708-343baa1d10da","order_by":1,"name":"Aisha Atnagulova","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Aisha","middleName":"","lastName":"Atnagulova","suffix":""},{"id":630499785,"identity":"e1f0abd9-7658-462a-b4bb-df01edc1a741","order_by":2,"name":"Dmitry Vorontsov","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Dmitry","middleName":"","lastName":"Vorontsov","suffix":""},{"id":630499789,"identity":"634138c1-b1ce-4548-83d3-6e6b11718a5a","order_by":3,"name":"Victoria Melnikova","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"","lastName":"Melnikova","suffix":""},{"id":630499791,"identity":"4b422557-40ac-409e-a62d-261fc3cc0c44","order_by":4,"name":"Yulia Nikishina","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Yulia","middleName":"","lastName":"Nikishina","suffix":""},{"id":630499793,"identity":"50589902-d7fa-4c93-8e08-325e6cf58ad2","order_by":5,"name":"Maxim Mezheritskiy","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Maxim","middleName":"","lastName":"Mezheritskiy","suffix":""},{"id":630499797,"identity":"03936546-6009-4819-bc99-e7ec0debbffd","order_by":6,"name":"Igor Zakharov","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Igor","middleName":"","lastName":"Zakharov","suffix":""},{"id":630499799,"identity":"a9d9f282-5e5f-410a-8de6-d1850760cb6d","order_by":7,"name":"Ilya Chistopolsky","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Ilya","middleName":"","lastName":"Chistopolsky","suffix":""},{"id":630499801,"identity":"4e80ba71-9814-45f3-b25a-796868bd8057","order_by":8,"name":"Angelina Akishina","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Angelina","middleName":"","lastName":"Akishina","suffix":""},{"id":630499806,"identity":"3e759e9b-f8b1-43ac-bf5e-eb3cc44c67b7","order_by":9,"name":"Varvara Dyakonova","email":"","orcid":"","institution":"Koltzov Institute of Developmental Biology","correspondingAuthor":false,"prefix":"","firstName":"Varvara","middleName":"","lastName":"Dyakonova","suffix":""}],"badges":[],"createdAt":"2026-04-01 11:09:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9290981/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9290981/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108014622,"identity":"cc542a0f-80ad-44a6-a617-1d503cef7fdd","added_by":"auto","created_at":"2026-04-28 13:32:20","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":648641,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in respiratory rate during two hours of intense locomotion in low water. A, \u003cem\u003eL. stagnalis\u003c/em\u003e with an open pneumostome (green arrow); B, scheme of apparatus for recording pneumostome states during crawling in low water. Symmetrically lit transparent arena was filled for 1 mm with water (marked with blue line), the breathing of a snail was recorded by camera located below; C, total time of pneumostome open state in the first and last 10 minutes of a two-hour training session (s); D, average duration of a single pneumostome open state in the first and last 10 minutes of a two-hour training session (s); F, number of respiratory cycles in the first and last 10 minutes of training. Asterisks indicate statistical difference, p\u0026lt;0.05, Wilcoxon test. F, Effect of acute exercise on the content of dopamine within central ganglia of \u003cem\u003eL.stagnalis\u003c/em\u003e, measured by HPLC\u003cem\u003e. \u003c/em\u003ePedal ganglia, grey bars; cerebral ganglia, white bars, control snails are shown by red circles, exercised snails are shown by green triangles. Significant decrease in the content of dopamine is detected after exercise in the pedal ganglia (p = 0.001).\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/2c2ca7f00895ccd49215e590.png"},{"id":108181045,"identity":"0328a215-0bb1-41d6-a985-7ae51e99a745","added_by":"auto","created_at":"2026-04-30 08:56:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":664591,"visible":true,"origin":"","legend":"\u003cp\u003ePedal ganglia of \u003cem\u003eLymnaea stagnalis\u003c/em\u003eand investigated neurons. A, Schematic representation of pedal ganglia, the serotonergic cluster of PeA neurons are marked with orange, identifiable neurons PeA2 and PeA8 are marked by arrows; the dopaminergic neuron RPeD1 is marked with green. B, RPeD1 in the pedal ganglia immunohistochemically stained by antibodies to tyrosine-hydroxylase the rate-limiting dopamine enzyme (ABClonal, China). At the same photo, the contours of unstained PeA cluster are also seen. CLSM, maximum intensity projection.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/d45738c808fc13842d3ffaa5.png"},{"id":108181170,"identity":"8df61292-5138-4e03-b2ee-5ab178c55fc7","added_by":"auto","created_at":"2026-04-30 08:58:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":512051,"visible":true,"origin":"","legend":"\u003cp\u003eThe response of dopaminergic neuron RPeD1 to single acute exercise for two hours. Membrane potential (A) in millivolts and spiking activity (B) in action potentials per minute of the dopaminergic neuron RPeD1 in control (n = 12) and exercised (n = 18) snails. Asterisks indicate statistical difference, U-test. C, example records of RPeD1 neuron in preparations of central ganglia dissected from control (upper trace) and exercised (lower trace) snail, located in the same dish. D, the same, one minute prior to and 10 minutes after administration of dopamine (DA, 0.05 mM) in snails after exercise (n = 8) and in the control (n = 8). E, F, membrane potential and spiking activity of RPeD1 prior to and after administration of dopamine, respectively. Asterisks indicate statistical difference, Wilcoxon paired test.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/ac6cd84b9803dfc324b8d5f9.png"},{"id":108014630,"identity":"352c5106-4a3c-45d8-9321-0c4270f04783","added_by":"auto","created_at":"2026-04-28 13:32:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":494765,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of chronic regular exercise on the baseline content of dopamine, l-DOPA (the precursor of dopamine) and serotonin in central ganglia of \u003cem\u003eL. stagnalis. \u003c/em\u003eMeasurements were taken the day after the final exercise experience, thus reflecting long-term changes in the central nervous system induced by exercise. Pedal ganglia, grey bars; cerebral ganglia, white bars; control snails data are shown in orange, the two-week regularly exercised snails (TWRE) are shown in olive. Significant decrease in the content of dopamine is detected after chronic exercise in the pedal ganglia (p = 0.038).\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/e1e7605e002986ea713e6d42.png"},{"id":108014624,"identity":"28cefc1b-6ea0-4c19-ad79-7ea3a6b49511","added_by":"auto","created_at":"2026-04-28 13:32:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":143674,"visible":true,"origin":"","legend":"\u003cp\u003eThe response of dopaminergic neuron RPeD1 to chronic regular exercise for two weeks. Measurements were taken the day after the final exercise, thus reflecting long-term changes in the central nervous system induced by physical activity. Changes in membrane potential (A) and spiking activity (B) of RPeD1 neuron in the isolated central ganglia preparations induced by chronic exercise. Asterisks indicate statistical difference, ** p\u0026lt;0.01. C, Example records of RPeD1 activity from the moment the electrode penetration in control and two-week regularly exercised (TWRE) snails.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/91d75cbcf5750eb449863038.png"},{"id":108181302,"identity":"a6eea5b0-9f8c-4ec3-bc0c-0b2d7cfbf425","added_by":"auto","created_at":"2026-04-30 08:58:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":214282,"visible":true,"origin":"","legend":"\u003cp\u003eThe response of the serotonergic neurons of PeA cluster to chronic regular exercise for two weeks. Measurements were taken the day after the final exercise, thus reflecting long-term changes in the central nervous system induced by physical activity. Changes in membrane potential (A) and spiking activity (B) of serotonergic PeA neurons in isolated central ganglia preparations induced by chronic exercise. C, Example records of PeA neuron activity of control snail and two-week regularly exercised (TWRE) snail during the isolation. Cells were first recorded within the CNS (left part of the trace), during mechanical isolation by pulling with the recording electrode (marked with color bar below the trace), and after complete isolation from the CNS (right part of the trace). E, F, changes in membrane potential and spiking activity of isolated serotonergic PeA neurons (measured ten minutes after the isolation of the neuron) induced by chronic exercise. Measurements were taken the day after the final exercise, thus reflecting long-term changes in the central nervous system induced by exercise. Asterisks indicate statistical difference,* p\u0026lt;0.05; ** p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/fa80ae4e1f7db2081027d57f.png"},{"id":108181855,"identity":"3d99c623-3c7a-4cf3-8f16-7fee39da4ed4","added_by":"auto","created_at":"2026-04-30 08:58:58","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":692790,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of chronic exercise on the speed of locomotion. A, Tracks of eight snails in low water arena; B, The mean speed of locomotion (pooled from four groups) during 2 hours of exercise in the fist, seventh and the 14th day of chronic regular exercise in low water. C, the dynamic of speed (mm/s) during two hours of exercise in four experimental groups of snails (marked with different colors) on the first day of training in low water; D, the dynamic of speed (mm/s) during two hours of exercise in four experimental groups of snails (marked with different colors) on the 14th day of training in low water. E, The effect of chronic exercise on the speed of locomotion in completely dry asymmetrically lit arena. The dynamics of locomotion speed (cm/s) during 15 min of behavior in novel completely dry asymmetrically lit arena of control (C, orange) and chronically exercised (TWRE, olive) snails. Measurements were taken the day after the final exercise.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/24c80d0a780ddff71dcf131a.png"},{"id":108014627,"identity":"2d2c40e2-86e6-4ff0-8fda-d62e0b9fe3a6","added_by":"auto","created_at":"2026-04-28 13:32:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":253952,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of regular exercise on the growth, consumption and oviposition in \u003cem\u003eL. stagnalis\u003c/em\u003e. A, The weight of control and exercised snails after two weeks; B, the mean weight of consumed food during two weeks, evaluated daily, in control and exercised snails. C, the number of egg clutches, evaluated daily, in control and exercised snails. Note the effect of water change at the days 1, 4, 8, 11, 14. D, the mean number of egg clutches in control and exercised snails.\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/ea6f0606f455aee3390dee96.png"},{"id":108184209,"identity":"27b72094-4f28-43db-9796-fb100eceb301","added_by":"auto","created_at":"2026-04-30 09:03:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3690349,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9290981/v1/d64a71cf-df42-4b9c-a98f-2038763bede2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Chronic exercise changes the baseline properties of central monoaminergic systems, identified neurons and related behaviors","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRegular physical exercise exerts a multifaceted effect on behavior and the body. Changes in the musculoskeletal system have been well studied. These changes are known to be determined by the type of exercise: short-term strength training leads to greater increases in muscle mass and strength, while longer-term aerobic exercise improves endurance, allowing adaptation to the training stimulus. Adaptation manifests itself in structural and metabolic changes of muscle fibers: short-term exercise can be provided by the energy derived from rapid anaerobic glycolytic breakdown of glucose, while prolonged exercise requires a slower but more efficient process of oxidative phosphorylation occurring in the mitochondria [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. At the same time, it is generally understood that changes in the muscular system are only the tip of the iceberg, while the main events that determine the impact of exercise on behavior occur in the brain [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Less is known about neuronal events accompanying exercise, especially with regard to individually identified neurons. Although it is known that exercise activates neurogenesis and influences neurotransmitter, neurotrophic, and neuromodulatory systems [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], as well as gene expression in the mammalian brain [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], it remains unclear how a specific neuron adapts its properties to acute or repeated physical activity. Recent work in mammals has begun to address this issue by demonstrating that repeated exercise enhances synaptic input strength and increases the intrinsic excitability of defined hypothalamic neurons (VMH SF1), and that the activity of these neurons is causally required for endurance adaptations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. These findings indicate that exercise-induced plasticity at the level of identified neuronal populations can directly mediate physiological adaptation, but the cellular mechanisms remain difficult to dissect in vertebrate systems.\u003c/p\u003e \u003cp\u003eThe relationship between presumed changes in specific neurons and subsequent changes in other behaviors, such as learning, decision-making, feeding, and reproduction, that are known to be influenced by acute and chronic exercise [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], remains to be elucidated. These questions are challenging to address in the complex mammalian brain. However, several simpler models have recently emerged for studying changes in the nervous system during exercise [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe previously found that, in the mollusc \u003cem\u003eL. stagnalis\u003c/em\u003e, a single two-hour experience of intense muscular crawling in low water facilitates proactive behaviors, decision-making in a novel environment [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and enhances reproduction [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Such exercise affected the expression of a number of genes in the snail nervous system [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Notably, dozens of homologous genes were also affected by exercise in the mouse brain [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. By taking advantage of the \u003cem\u003eLymnaea\u003c/em\u003e model for cellular analysis, we previously reported the accompanying changes in the membrane potential and the activity of identified central serotonergic neurons involved in locomotion [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These changes persisted even after the complete isolation of the neurons from the central ganglia. In addition, we obtained evidence for changes in the dopaminergic system following exercise (17). In \u003cem\u003eLymnaea\u003c/em\u003e, the involvement of dopaminergic mechanisms in exercise is plausible since the giant pedal dopaminergic neuron RPeD1 is a component of the respiratory central pattern generator (CPG) in \u003cem\u003eL. stagnalis\u003c/em\u003e, while an increased respiratory rate accompanies exercise in most animals [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe aim of this study was to test the hypothesis that monoaminergic neurons develop a state of readiness for exercise during chronic intense locomotion by changing their baseline properties. To test this, we used the same \u003cem\u003eLymnaea\u003c/em\u003e model and compared the activity of identified monoaminergic neurons and brain monoamine levels immediately after a single acute two-hour exercise and one day after the end of the two-week daily two-hour exercise protocol. To analyze the neuronal effects of exercise, we selected serotonergic neurons of the PeA cluster that directly control locomotion [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and the unpaired dopaminergic neuron RPeD1, which is involved in the generation of the respiratory rhythm [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, we recorded and analyzed changes in the locomotor, feeding, and reproductive behaviors during chronic regular exercise.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEffects of single acute exercise\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eExercise enhances breathing in L. stagnalis\u003c/h2\u003e \u003cp\u003eAn increased breathing rate accompanies intense locomotion in most animals studied in this context [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. We tested whether the same occurs during intense crawling in \u003cem\u003eL. stagnalis\u003c/em\u003e. The mean and the total duration of pneumostome openings (PNO, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), summed over 10 min, were significantly higher during the last 10 minutes compared to the first 10 minutes of the 2-h locomotor session (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D, n\u0026thinsp;=\u0026thinsp;20, Wilcoxon test: p\u0026thinsp;=\u0026thinsp;0.02, 0.027, correspondingly). The number of PNOs was significantly higher at the end of the exercise (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE; n\u0026thinsp;=\u0026thinsp;20; paired Wilcoxon signed-rank test: p\u0026thinsp;=\u0026thinsp;0.01). Thus, breathing rate increased during 2 hours of intense locomotion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the control group (n\u0026thinsp;=\u0026thinsp;20, no differences were detected in PNO frequency or duration between the beginning and end of the same 2-h interval (Wilcoxon test for differences in opening frequency in the control: p\u0026thinsp;=\u0026thinsp;0.328; for total duration of open state of pneumostome: p\u0026thinsp;=\u0026thinsp;0.1; and for average duration: p\u0026thinsp;=\u0026thinsp;0.13). This is consistent with the increased locomotion speed observed after two hours of low-water crawling in \u003cem\u003eL. stagnalis\u003c/em\u003e [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and with the previously reported coordination of respiratory and locomotor rhythms [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eAcute exercise decreases dopamine content in the pedal ganglia\u003c/h3\u003e\n\u003cp\u003eThe dopamine (DA) content in the pedal ganglia decreased significantly immediately after the acute 2-h exercise (t-test, p\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, the left group of bars). In contrast, DA content in the cerebral ganglia did not differ between control and exercised snails (Mann\u0026ndash;Whitney U Statistic\u0026thinsp;=\u0026thinsp;15; p\u0026thinsp;=\u0026thinsp;0.699, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, the right group of bars).\u003c/p\u003e \u003cp\u003eTwo hours of rest in deep water after the acute exercise abolished the decrease of DA in pedal ganglia. DA content was 82.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5 pmol in exercised-then-rested snails (n\u0026thinsp;=\u0026thinsp;6) and 67.6\u0026thinsp;\u0026plusmn;\u0026thinsp;5.2 pmol in control snails (n\u0026thinsp;=\u0026thinsp;6) kept for four hours in deep water and handled twice to match the exercised-then-rested group. A non-significant trend toward a rebound increase of DA content was observed in exercised-then-rested snails (p\u0026thinsp;=\u0026thinsp;0.093). Thus, the exercise-induced changes in DA content were transient and were not maintained after a single exercise bout.\u003c/p\u003e\n\u003ch3\u003eAcute exercise decreases the tonic activity of the dopaminergic RPeD1 neuron\u003c/h3\u003e\n\u003cp\u003eRPeD1 neuron (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B) is known to have several modes of activity. It can generate spikes tonically in the absence of respiratory rhythm. It is thought that sensory cells in the pneumostome-osphradium region, activated by chemosensory stimuli (e.g., hypoxia), provide strong excitatory afferent input to RPeD1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Through synaptic connections within the respiratory CPG, activated RPeD1 initiates CPG activity underlying the respiratory rhythm. During respiratory CPG activity, RPeD1 becomes hyperpolarized, ceases tonic firing, and exhibits rhythmic bursting driven by EPSP bursts from respiratory CPG interneurons [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter intense locomotion, RPeD1 neurons (n\u0026thinsp;=\u0026thinsp;18) were in a more hyperpolarized state (Mann-Whitney U-test: p\u0026thinsp;=\u0026thinsp;0.013, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and exhibited significantly lower spike frequency (Mann-Whitney U-test: p\u0026thinsp;=\u0026thinsp;0.007, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB,С) than neurons from control snails (n\u0026thinsp;=\u0026thinsp;18) recorded in parallel.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eThe hyperpolarizing effect of acute exercise on RPeD1 cannot be explained by reduced dopamine content of in the pedal ganglia\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe giant dopaminergic RPeD1 neuron expresses dopamine receptors [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Given this finding, reduced DA content in the pedal ganglia of exercised snails could influence the spiking activity and membrane potential of RPeD1. To test this, we examined the effect of exogenous DA on RPeD1 neurons in the CNS isolated from control and exercised snails.\u003c/p\u003e \u003cp\u003eIn both control (n\u0026thinsp;=\u0026thinsp;8) and exercised (n\u0026thinsp;=\u0026thinsp;8) snails, DA hyperpolarized RPeD1 neurons (Fig.\u0026nbsp;3С-F). After DA application, the difference between preparations from control and exercised snails became even more evident: DA completely suppressed tonic firing of RPeD1 from exercised snails, whereas tonic activity persisted in control preparations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This difference might, in part, reflect pre-existing baseline differences between the groups. Nevertheless, the possibility of an elevated sensitivity of RPeD1 to DA (and potentially enhanced autoinhibitory feedback via DA receptors) in preparations from exercised snails cannot be dismissed. Taken together, these findings suggest that the post-exercise hyperpolarization of RPeD1 is not a consequence of reduced DA concentrations within the pedal ganglia. Rather, it may reflect training-induced alterations in intrinsic membrane properties that set the resting membrane potential and/or an increased sensitivity to dopamine\u0026rsquo;s hyperpolarizing action.\u003c/p\u003e\n\u003ch3\u003eEffects of chronic exercise\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTwo-week regular exercise decreased baseline DA content in the pedal ganglia\u003c/h2\u003e \u003cp\u003eWe measured dopamine content 24 h after the final session of the two-week daily exercise protocol. Baseline DA levels in the pedal ganglia were significantly reduced compared with controls (n\u0026thinsp;=\u0026thinsp;12 per group, p\u0026thinsp;=\u0026thinsp;0.039; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, right panel). In contrast, levels of the DA precursor L-3,4-dihydroxyphenylalanine (L-DOPA) did not differ between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). This pattern is consistent with increased DA turnover rather than reduced synthesis, although synthesis and degradation were not directly measured. No significant changes were detected in the cerebral ganglia for either DA or L-DOPA. Thus, chronic exercise affected pedal ganglia DA in the same direction as acute exercise.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChronic exercise for two weeks decreased baseline activity of dopaminergic RPeD1 neuron\u003c/h3\u003e\n\u003cp\u003eAfter the two-week exercise protocol, RPeD1 was hyperpolarized (n\u0026thinsp;=\u0026thinsp;11 per group, p\u0026thinsp;=\u0026thinsp;0.007; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), and its firing activity was significantly reduced (n\u0026thinsp;=\u0026thinsp;11 per group, p\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C); in most preparations, the neuron was silent. This state resembled the response observed after a single 2-h acute exercise bout.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eChronic exercise for two weeks did not alter baseline serotonin levels\u003c/h3\u003e\n\u003cp\u003eThe effect of a single acute exercise bout on serotonin (5-HT) and its catabolites in the pedal and cerebral ganglia have been described previously [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Both ganglia contain numerous serotonergic neurons, including those involved in locomotion. Acute exercise did not significantly change 5-HT levels; however, during the post-exercise rest period, levels of all measured serotonin catabolites were significantly increased [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Here we examined whether chronic exercise affects serotonin levels. No significant changes were detected in either the cerebral ganglia or the pedal ganglia after the two-week exercise protocol (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). In the pedal ganglia, there was a trend toward decreased 5-HT levels (p\u0026thinsp;=\u0026thinsp;0.06).\u003c/p\u003e \u003cp\u003e \u003cem\u003eChronic exercise for two weeks induced depolarization of serotonergic PeA cluster neurons in situ and after complete isolation\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe effect of single acute exercise on electrical properties of serotonergic PeA-cluster neurons in situ (within the CNS) and after complete isolation have been described earlier [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Depolarization and higher spiking activity were observed in these cells after acute exercise, which agreed with behaviorally observed increase in the locomotion speed [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfter chronic exercise, the serotonergic neurons of PeA cluster were depolarized (-48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.98 mV versus \u0026minus;\u0026thinsp;54.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3 mV for control; n\u0026thinsp;=\u0026thinsp;38, 33; t\u0026thinsp;=\u0026thinsp;3.34, p\u0026thinsp;=\u0026thinsp;0.0013, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA), but their spiking activity remained virtually unchanged (40.2\u0026thinsp;\u0026plusmn;\u0026thinsp;9.4 AP/min versus 30.9\u0026thinsp;\u0026plusmn;\u0026thinsp;6.4 AP/min for control; t\u0026thinsp;=\u0026thinsp;1.6; p\u0026thinsp;=\u0026thinsp;0.1, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter complete isolation of the PeA cluster neurons from the CNS (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, D), group differences became more pronounced. In chronically exercised snails, isolated serotonergic neurons demonstrated significant depolarization (-46.6\u0026thinsp;\u0026plusmn;\u0026thinsp;4.4 mV versus \u0026minus;\u0026thinsp;55.3\u0026thinsp;\u0026plusmn;\u0026thinsp;5.8 for control; n\u0026thinsp;=\u0026thinsp;18, 19; Mann-Whitney U-test z\u0026thinsp;=\u0026thinsp;2.3, p\u0026thinsp;=\u0026thinsp;0.019, (normality test p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE) and higher spiking rate (34.9\u0026thinsp;\u0026plusmn;\u0026thinsp;9.6 AP/min versus 16.7\u0026thinsp;\u0026plusmn;\u0026thinsp;9.5 AP/min for control; Mann-Whitney U-test z\u0026thinsp;=\u0026thinsp;2.7, p\u0026thinsp;=\u0026thinsp;0.007, (normality test p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). These effects resemble those previously reported after a single acute exercise bout [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral changes produced by two-week chronic exercise\u003c/h2\u003e \u003cp\u003eLocomotion speed was analyzed separately in each of the four experimental groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, C, D). For statistical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB), data from four groups were pooled. Mean locomotion speed was significantly increased after one week of exercise compared with the first day (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). On the final day of two-week protocol, the increase in speed was smaller than after one week but remained statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The within-session dynamics of locomotion speed during the 2-h training period also differed on the final day compared with the first day (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD compare to Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). On the first day, locomotion started at a lower speed and increased toward the end of the session. In contrast, on the final day animals exhibited a significantly higher initial speed, which progressively decreased over the session.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLocomotion in a completely dry arena\u003c/h2\u003e \u003cp\u003eWe also tested snail behavior in a completely dry, asymmetrically lit arena [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] one day after the end of the two-week exercise protocol, i.e., under the same conditions as those used for electrophysiological and HPLC experiments. Trained snails exhibited higher speeds than control snails, as shown in the Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE ((n\u0026thinsp;=\u0026thinsp;12 per group) Wilcoxon paired test for means at each time interval, p\u0026thinsp;=\u0026thinsp;0.005). This result is consistent with the increased locomotion speed observed during training in low water. However, no difference was found in any other behavioral parameters between control and experimental snails, including the number of orienting turns (p\u0026thinsp;=\u0026thinsp;0.4, t-test, normal distribution) or time to the border of the arena (p\u0026thinsp;=\u0026thinsp;0.33, U-test). The dispersion of some parameters was lower in the exercised group, such as the time spent in the central zone (Fligner p value for equal dispersion 0.005).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWeight and growth\u003c/h2\u003e \u003cp\u003eSignificant differences in body mass were detected after the two-week chronic exercise protocol. Exercised snails weighed less than control (1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14 g versus 2.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 g in the control group (t\u0026thinsp;=\u0026thinsp;4; p\u0026thinsp;=\u0026thinsp;0.0003, (normality test p\u0026thinsp;\u0026gt;\u0026thinsp;0.1), Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003ePossible impact of weight difference on locomotion speed in experimental and control animals\u003c/h2\u003e \u003cp\u003eTo test the possible influence of weight differences on the speed of locomotion in experimental and control snails, we compared the behavior of intact large (5.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5g) and small (1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3g) animals in the same dry arena. There was no significant difference between small and large snails in any of the evaluated parameters: distance, velocity, time spent in the center, first exit, number of turns (p\u0026thinsp;\u0026gt;\u0026thinsp;0.09). The larger snails showed a trend for a higher speed of locomotion (0,054 cm/s versus 0,045 cm/s; p\u0026thinsp;=\u0026thinsp;0.0952), which is opposite to the difference observed between control and TWRE snails. Thus, we conclude that the difference in body weight does not explain the higher speed of locomotion observed in chronically exercised snails.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eConsumption\u003c/h2\u003e \u003cp\u003eTo determine whether reduced growth could be explained by lower food intake, consumption was measured in an additional experimental series. Contrary to this hypothesis, food intake was higher in the exercised group throughout the 13-days observation period. The mean mass of consumed food was 3.13 g in control versus 4.7 g in exercised snails (n\u0026thinsp;=\u0026thinsp;13; Z\u0026thinsp;=\u0026thinsp;2.9, p\u0026thinsp;=\u0026thinsp;0.0037 paired Wilcoxon test, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eOviposition\u003c/h2\u003e \u003cp\u003eClean water is known to stimulate egg laying in \u003cem\u003eL. stagnalis\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. This effect was evident throughout the entire chronic exercise experiment, when the water was replaced in containers of both control and exercised groups (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, days 1, 4, 8, 11, 14). The number of egg clutches was higher but only as tendency (t-test, p\u0026thinsp;=\u0026thinsp;0.05) in the exercised group (Fig.\u0026nbsp;9D). Average number of eggs over two weeks was 746.5\u0026thinsp;\u0026plusmn;\u0026thinsp;80 in control and 1006.5\u0026thinsp;\u0026plusmn;\u0026thinsp;55 in exercised snails. The number of eggs per clutch did not differ significantly (t-test, p\u0026thinsp;=\u0026thinsp;0.09).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe primary aim of this study was to test the hypothesis that monoaminergic neurons develop a state of readiness for exercise during regular intense locomotion by altering their baseline properties. Towards this aim, we compare the activity of identified neurons involved in locomotion and respiration, as well as monoamine levels, immediately after a single 2-h exercise session and 24 h after completing a two-week daily exercise protocol.\u003c/p\u003e \u003cp\u003eWe observe similar changes in monoaminergic neuron state and ganglionic monoamine content immediately after a single exercise bout and 24 h after two weeks of daily exercise. In both cases, the serotonergic neurons of PeA cluster, which are related to locomotion, were depolarized, both \u003cem\u003ein situ\u003c/em\u003e and after their complete isolation, while no significant changes were observed in the serotonin content. At the same time, the respiration-related dopaminergic neuron RPeD1 was hyperpolarized and dopamine content was decreased in both cases. These findings strongly support the idea that baseline properties of monoaminergic systems are affected by regular exercise. The similarity of these changes with the effects of acute exercise may suggest an anticipatory/predictive mechanism. Notably, post-training measurements were performed at the time of day when the next exercise session would normally occur. We argue against a single-switch explanation in which the first exercise bout produces a persistent state change. Consistent with this, a 2-h rest period after a single acute exercise bout produces changes opposite to those observed immediately after exercise [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe great pond snail \u003cem\u003eL. stagnalis\u003c/em\u003e is a relatively new model for studying the effects of exercise on the brain. Importantly, this model incorporates an ethologically and ecologically relevant behavioral paradigm. The form of intense locomotion, which is treated here as exercise, is specific for this snail. It is activated when the snail is in shallow water or actively crawls onto the land. \u003cem\u003eL.stagnalis\u003c/em\u003e has already been shown to share many similarities with mammals in the behavioral effects of exercise, such as reduced fearfulness, increased activity and determination in a new environment, facilitated decision-making [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarities with mammals also include the involvement of monoaminergic systems in these effects [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and are present even at the level of gene expression: in our recent study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], we discovered a non-random overlap of gene subsets whose brain expression is influenced by exercise both in mice [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and snails. A number of similar functional clusters of genes responded to exercise in these distinctly-related species [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOne of the main advantages of \u003cem\u003eL. stagnalis\u003c/em\u003e as a model animal is that it allows tracing the entire chain of effects associated with exercise in the central nervous system, starting from the very first stage \u0026ndash; changes in the properties of central serotonergic neurons of the PeA cluster, which send projections to the sole and to the pedal cilia and thus directly regulate locomotion [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. So far, the shift in the individual biophysical characteristics of identified neurons caused by exercise has been directly demonstrated only in \u003cem\u003eLymnaea\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, changes in the membrane potential are detected in serotonergic neurons of the PeA cluster and in the dopaminergic neuron RPeD1 both immediately after a single acute exercise and after two weeks of chronic exercise. In this regard, it should be noted that the expression level of the UNC80 gene, which plays a role in regulating membrane potential, was affected by exercise in both mice and snails [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Unc80 is an evolutionarily conserved gene. It encodes a protein that is a component of the voltage-independent leakage ion channel complex, which plays an important role in establishing resting membrane potential in neurons of both invertebrates and mammals [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. It is widely expressed in the nervous system of the nematode \u003cem\u003eC. elegans\u003c/em\u003e, and worms with the knockout of the Unc80 gene are capable of crawling but are unable to switch to more intense and rapid locomotion, such as swimming [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In humans, mutations in Unc80 are associated with congenital infantile encephalopathy, mental retardation, and growth problems [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. We hypothesize that the membrane potential of some key neurons related to locomotion is affected by exercise not only in the mollusk, where we directly demonstrate this, but also in the mammalian brain, where it still remains challenging to track changes in the biophysical properties of individual neurons. This is consistent with the exercise-induced Unc80 expression changes reported in the mammalian brain. Our assumption is supported by recent evidence showing that repeated exercise increases the excitability of VMH SF1 neurons in mice, and that suppression of their post-exercise activity prevents the development of endurance adaptations [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Thus, exercise-induced shifts in baseline neuronal excitability appear to represent a conserved mechanism contributing to systemic effects of training.\u003c/p\u003e \u003cp\u003eThe possible role of 5-HT in the modulating effects of exercise has been investigated and discussed earlier [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, an increased release of 5-HT can explain only some of the known effects of intense locomotion in \u003cem\u003eL. stagnalis\u003c/em\u003e. Serotonin increases the speed of locomotion in novel environment and can contribute to the higher oviposition rate observed after a single exercise [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. However, elevated 5-HT levels do not explain changes in goal-oriented behavior and decision-making [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we initiated a cellular analysis of the potential involvement of respiratory system neurons in mediating the effects of exercise. It is generally known that intense physical activity has a significant impact on the respiratory system because of the close relationship between body movements, oxygen demand and breathing. One of the main mechanisms of this interaction is the coordination of breathing rhythms with walking or running. For example, recent research has shown that a specialized region of the brain called the mesencephalic locomotor area can activate respiratory centers in advance, before the blood carbon dioxide levels rise [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. That discovery suggests that an organism increases breathing ahead of exercise, preparing itself for increased physical exertion, and that central neural control of respiration demonstrates significant autonomy from peripheral chemoreceptors [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Of importance is the locomotor-respiratory coordination. In humans, breathing is frequently synchronized with the stepping rhythm, reducing overall energy expenditure [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Such coordination has also been demonstrated in \u003cem\u003eL. stagnalis\u003c/em\u003e, which possesses neurons that simultaneously regulate respiration and intense locomotion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. One such neuron investigated here is RPeD1 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Immunohistochemical studies have shown that this neuron contains tyrosine hydroxylase (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), confirming its dopaminergic nature [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. RPeD1 projects into the right and left pedal ganglia and also sends axons to the cerebral and pleural ganglia via connecting nerves, forming synaptic connections with other neurons involved in the regulation of respiration and locomotion [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Neurons displaying D1 receptor-like immunoreactivity have been observed in all ganglia of \u003cem\u003eL. stagnalis\u003c/em\u003e but particularly in the pedal ones [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It should be noted that, functionally, the Lymnaea pedal dopaminergic system is involved not only in the regulation of respiratory rhythm [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Thus, studies have shown that dopaminergic neurons, including RPeD1, play a key role in the formation of long-term memory, particularly in the context of operant breathing learning [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Dopaminergic neurons are also present in various peripheral tissues [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], peripheral sensory dopaminergic axons innervate the pedal ganglia in molluscs [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In the pedal cilia of \u003cem\u003eL.stagnalis\u003c/em\u003e, dopamine exerts the effect opposite to serotonin, namely, inhibits cilia beating and decreases the speed of locomotion [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere we show that respiratory rhythm indeed increases toward the end of a two-hour exercise in the \u003cem\u003eL.stagnalis\u003c/em\u003e. Surprisingly, the RPeD1 neuron, which is believed to initiate and maintain the respiratory rhythm generation, was hyperpolarized in trained snails. As was mentioned above, this neuron exhibits diverse activity patterns. In the absence of a respiratory rhythm, it can fire action potentials continuously. It is hypothesized that sensory cells located in the pneumostome-osphradium region, when stimulated by chemosensory cues such as hypoxia, deliver robust excitatory afferent input to the RPeD1 neuron [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This activation of RPeD1 is thought to trigger the CPG activity that drives the respiratory rhythm. Nevertheless, during the active generation of the respiratory CPG, the RPeD1 neuron itself undergoes hyperpolarization. This hyperpolarization suppresses its intrinsic tonic firing, causing it to display rhythmic bursting activity instead, which is driven by incoming bursts of excitatory postsynaptic potentials (EPSPs) originating from the respiratory CPG interneurons [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The same hyperpolarizing effect is produced by dopamine as we show here and also earlier [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In the latter study, the inhibiting effect of dopamine on autoreceptors of RPeD1 is confirmed in completely isolated RPeD1 neurons. Thus, the hyperpolarized state observed in trained snails could reflect the state of RPeD1 during respiratory rhythm generation. However, this effect cannot be explained by reduced dopamine levels in the pedal ganglia after exercise, because dopamine itself hyperpolarizes RPeD1. Instead, it appears to be a consequence of alterations in intrinsic mechanisms governing membrane potential, and/or a substantially elevated responsiveness to dopamine's hyperpolarizing influence.\u003c/p\u003e \u003cp\u003eNotably, the same combination of effects (decreased dopamine content and hyperpolarized membrane potential of RPeD1) was observed after two weeks of regular exercise, but after 24 hours since the last exercise episode. Chronic exercise not only altered the baseline properties of RPeD1 but also made them resemble the acute exercise state even 24 h after the last session. This finding agrees with the idea that \u0026ldquo;organism increases breathing in advance, preparing itself for increased physical exertion\u0026rdquo; [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterestingly, this neuronal \u0026ldquo;preparedness\u0026rdquo; is consistent with the increased locomotion speed observed in chronically exercised snails. By the end of the second week, locomotion speed remained elevated relative to day 1. The overall speed profile changed over the two hours of exercise: while, on the first day, the animals started at comparatively low speed and finished at higher speed, on the last day of chronic exercise, the opposite pattern was observed: a high start and a low finish. It is possible that the exercise protocol that we chose was somewhat excessive, which was reflected in a reduced average speed after 14 days compared to seven days of exercise, as well as in the progressive slowing over two hours of exercise observed on the last day. The behavior of chronically exercised and control snails in novel, completely dry conditions confirmed the ability of trained snails to develop faster locomotion even under unfamiliar stressful conditions. This effect occurred even though they had a lower body mass than controls.\u003c/p\u003e \u003cp\u003eMonoaminergic systems are known to affect other behaviors in \u003cem\u003eL. stagnalis\u003c/em\u003e, such as feeding and reproduction [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition, we propose that there may be other parameters beyond the monoamine levels and the activity of monoaminergic neurons that undergo changes during the exercise. To address this, during the two-week chronic exercise, in addition to locomotion we analyzed several other behaviors and states of the snails. We found that exercised snails exhibited higher food consumption and laid more eggs. However, their growth apparently slowed compared to the control group.\u003c/p\u003e \u003cp\u003eIncreased locomotor, feeding, and reproductive activities are consistent with the known effects of metabolic intensification induced by intense locomotion in other organisms, vertebrates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and invertebrates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Interestingly, in \u003cem\u003eL. stagnalis\u003c/em\u003e these effects were observed also after single exercise session [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Meanwhile, the significant inhibition of growth that compensated for these activities suggests the possible involvement of the brain insulin signaling in this shift of organismal state. Indeed, in many animals, neuronally-produced insulin determines the balance between energy expenditure, reproduction, and growth [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In \u003cem\u003eL. stagnalis\u003c/em\u003e, removal of specific brain regions, lateral lobes, containing insulin neurons results in the decrease in the mollusk's size [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] and activates reproduction [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The suppression of body growth appears to have been connected to the boosting of female reproductive output. Given that the consistent generation of egg masses, intense locomotion and the expansion of body size necessitate significant resource and energy expenditure, it's probable that some of these functions are mutually exclusive. Changes in several behaviors besides locomotion that we observed after exercise suggest that other neurons, probably related to feeding and reproduction, as well as other neuromodulatory systems, such as the insulin system, can similarly adjust their properties to the training.\u003c/p\u003e \u003cp\u003eIn conclusion, we present a new invertebrate model for studying the effects and mechanisms of chronic exercise. We show that behavioral effects of regular training in \u003cem\u003eL. stagnalis\u003c/em\u003e can be traced to changes in the properties of identified central neurons. Our results support the idea that regular exercise induces an anticipatory adjustment of baseline properties in monoaminergic neurons.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMature specimens of \u003cem\u003eLymnaea stagnalis\u003c/em\u003e were taken from a breeding colony kept in dechlorinated tap water at room temperature and regularly fed with lettuce ad libitum. Three-month-old snails weighing 1.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2 g were used for the experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral experiments\u003c/h2\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003eAcute exercise\u003c/h2\u003e \u003cp\u003eExperimental snails were placed for two hours in flat white trays, 25 x 50 cm, filled with 1 cm-deep water, taken from the colony aquarium as in [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Under these conditions, terrestrial locomotion (crawling) is induced, which is energetically more demanding than underwater ciliary locomotion. In parallel, control snails were removed from the domestic aquarium and placed into a new container filled with water from the colony aquarium where they could drift normally. After two hours, experimental and control snails were dissected under MgCl\u003csub\u003e2\u003c/sub\u003e 0.1 M anesthesia for electrophysiology or HPLC analysis of monoamine content. In an additional control series, the experimental animals were allowed to rest in deep water for two hours after exercise, and then were dissected for HPLC analysis.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of respiratory behavior during two hours exercise\u003c/h2\u003e \u003cp\u003eThe number and duration of pneumostome openings (PNO, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) were recorded during the first and last 10 minutes of the two-hour locomotor activity. A video camera positioned beneath the transparent floor of the arena allowed continuous recording of PNOs during locomotion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). For each snail, the frequency of PNOs was calculated from the video recordings based on these time intervals.\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eChronic exercise\u003c/h2\u003e \u003cp\u003ePrior to the experiment, we prepared eight groups of snails (n\u0026thinsp;=\u0026thinsp;8 per group) of the same size (10 weeks old). Animals were taken from their home aquarium and placed into 2.5 L containers (one group per container) filled with clean settled water and fed with lettuce ad libitum. During two days of adaptation to these novel conditions, we evaluated oviposition in all groups (the number of clutches and individual eggs) to exclude initial differences in these parameters between the groups. The groups were then assigned to either the experimental or control protocol.\u003c/p\u003e \u003cp\u003eFour experimental groups (eight snails per group) were placed in flat white trays (25 \u0026times; 50 cm) filled with 1 cm-deep water (one group per tray). After 2 hours of crawling in low water, the experimental groups were returned to their containers until the next day. In parallel, four groups of control snails were handled and returned to their containers. The same procedure was repeated daily at the same time (12:00) for two weeks. Every fourth day, the water in all containers was replaced with fresh water.\u003c/p\u003e \u003cp\u003eCrawling snails were video-recorded from above at 2 frames per second. The resulting videos were pre-processed in VirtualDub and then automatically tracked in FIJI [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] using the TrackMate plugin [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral test on the dry surface\u003c/h2\u003e \u003cp\u003eOne day after the end of the two-week exercise protocol, we compared the behavior of trained (two-week regular exercise, TWRE) and control snails under new conditions, in a completely dry arena as in [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This experiment was conducted to test the hypothesis that regular exercise under low water conditions pre-adapts snails to completely dry conditions. Individual snails (one at a time) were placed into a rectangular arena (60 x 45 cm) on a flat, dry plastic surface that aimed to simulate the potentially desiccating condition of being out of water (as in 13, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In such a situation, a snail is expected to select a direction of movement as quickly as possible to return to an aquatic environment and to avoid desiccation. One of the shorter walls of the arena was made of translucent plastic to provide the asymmetric illumination. All other walls were made of black opaque plastic. A white LED lamp was placed to the translucent side of the arena as a source of light. The light intensity at the arena\u0026rsquo;s center (measured 3 cm above the surface with the Proskit MT-4017 luxmeter) was made equal to 80 lux, and it was 120 and 20 lux near the light and the dark walls, respectively. No measurable amount of light came to the arena from below.\u003c/p\u003e \u003cp\u003eThe movements of each snail were recorded at 15 frames per second for 15 minutes with a video camera placed below the transparent plastic bottom of the arena. The recordings were tracked using the EthoVision XT software (Noldus, the Netherlands) and independently scored manually with RealTimer (Openscience, Russia). Snail turns were counted manually. The traces left by the crawling snails were removed with a clean paper towel before each new test.\u003c/p\u003e \u003cp\u003eDuring the analysis, a centered circle zone (10 x 10 cm) that limited the track and scoring analysis was defined. This zone was used to exclude from the analysis a snail's movements near the physical borders of the arena, especially near the brightly lit wall. We evaluated (i) mean locomotion velocity over 1-min intervals (ii) the number of rotations performed by the snail (iii) the time taken to crawl to the virtual border of the arena as in [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. When a snail did not reach the virtual border, the time was given the value of the total time of observation, which was 15 minutes. Manual scoring was performed blind to group assignment.\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eAnalysis of oviposition\u003c/h2\u003e \u003cp\u003e24 hours following each exercise session, the number of egg clutches, the number of eggs per clutch and the total number of eggs laid in the exercised and control groups were counted.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eAnalysis of consumption\u003c/h2\u003e \u003cp\u003eIn an additional experiment with two cohorts of snails (n\u0026thinsp;=\u0026thinsp;16 for control and n\u0026thinsp;=\u0026thinsp;16 for experiment) we controlled feeding behavior during the two-week daily exercise. The procedure of exercise was the same as described above.\u003c/p\u003e \u003cp\u003eEach day, immediately after the exercise session, each experimental and control group of snails received 5 g of fresh lettuce. After 12 hours, the remaining lettuce was removed from the containers and weighed. The consumed mass was calculated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eWeight gain analysis\u003c/h2\u003e \u003cp\u003eBoth the control and exercised snails were weighed after the two-week chronic exercise. The distribution was tested for normality, and then the significance of differences between groups was evaluated by t-test.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eElectrophysiology and cell isolation\u003c/h2\u003e \u003cp\u003eIn each experiment, two snails (experimental and control) were anesthetized by injection of 0.1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, followed by dissection of the central ganglia (central nervous system, CNS). Both CNS preparations were placed into a 3 mg/ml solution of pronase E (Sigma) for 15 min, washed in a standard snail Ringer\u0026rsquo;s solution (50 mM NaCl, 1.6 mM KCl, 4 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 8 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 10 mM Tris, pH 7.6) and then both CNS were pinned in a 40 mm Sylgard-bottomed chamber at a distance of approximately 10 mm between them. The connective tissue sheath was then removed from the pedal ganglia in both CNS.\u003c/p\u003e \u003cp\u003eVisual identification of the RPeD1 and PeA neurons was performed based on their location, size and coloration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). The neuron selected for recording was impaled with a standard glass microelectrode (10\u0026ndash;20 MΩ) filled with 3 M KCl. A standard setup for microelectrode recording was used. The electrophysiological recordings were stored in computer files using a home-made software.\u003c/p\u003e \u003cp\u003eMechanical isolation of a neuron was performed according to the previously developed method [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Using the intracellular microelectrode as a handle, the neuron was gently pulled out of the ganglion tissue until separation of the proximal neurite from the neuropile was achieved. The electrical activity of the cell was continuously monitored during isolation. The cells, which demonstrated membrane injury, were excluded from the following experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003eHigh Performance Liquid Chromatography\u003c/h2\u003e \u003cp\u003eCentral ganglia were quickly dissected on ice, homogenized using an ultrasonic homogenizer (Bandelin Sonopuls, Burladingen, Germany) at 4\u0026deg;C in 0.1 M HClO₄, and centrifuged at 10,000 \u0026times; g for 20 min at 4\u0026deg;C. The supernatant was collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C prior to monoamine quantification.\u003c/p\u003e \u003cp\u003eAn Agilent 1260 Infinity II HPLC system (Agilent Technologies Inc., Waldbronn, Germany), equipped with a fluorescence detector (FLD), was used for monoamine analysis. Analytes were separated using a reverse-phase InfinityLab Poroshell 120 EC-C18 column (100 mm \u0026times; 4.6 mm, 2.7 \u0026micro;m particle size; Agilent Technologies Inc., Germany). The column was thermostated at 30\u0026deg;C. The mobile phase consisted of 0.1 M citrate\u0026ndash;phosphate buffer, 0.25 mM sodium 1-octanesulfonate, 0.1 M EDTA, and 7% acetonitrile (pH\u0026thinsp;=\u0026thinsp;2.56) (all reagents purchased from Sigma-Aldrich, St. Louis, MO, USA). The mobile phase flow rate was 1 mL/min. FLD detection was carried out at an excitation wavelength of 285 nm, and emission was recorded at 310 nm. Peaks were identified based on retention times relative to standard solutions, and monoamine concentrations were calculated from the ratio of peak areas relative to the standards.\u003c/p\u003e \u003cp\u003eIn some experiments (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), DA, DOPA, and 5-HT were quantified using an electrochemical detector (Decade Elite, Antec, the Netherlands) equipped with a glassy carbon flow cell and a salt-bridge Ag/AgCl reference electrode, with the potential set at +\u0026thinsp;0.85 V.\u003c/p\u003e \u003cp\u003eMonoamine content is presented as pmol per ganglion, as the ganglia are very small and weighing the samples would increase measurement error.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eNormality of the data was assessed prior to statistical testing. Depending on the distribution, either the Mann\u0026ndash;Whitney U test or the Student\u0026rsquo;s t-test was used to evaluate differences between independent groups. For dependent samples, the paired Wilcoxon signed-rank test was applied.\u003c/p\u003e \u003cp\u003eStatistical analysis and figure preparation were performed using PAST [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] or the R Project for Statistical Computing. For the HPLC data, statistical analysis was performed using GraphPad Prism version 8.1.1.\u003c/p\u003e \u003cp\u003eAll values are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e Conceptualization, V.D. and D.V.; investigation, A.S., A.A., I.Ch., V.M., Yu.N., An. A., I.Z. and M.M.; writing\u0026mdash;original draft preparation, V.D.; writing\u0026mdash;review and editing, D.V., A.A. and A.S.; visualization, V.D., D.V. and A.S.; project administration, V.D. and I.S. All authors have read and agreed to the published version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e\u0026nbsp; Supported by RSF 25-14-00147.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis research was conducted using the equipment of the Core Centrum of the Institute of Developmental Biology RAS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data that support the findings of this study are available within the article. Further information can be obtained from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u003c/strong\u003e The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSmith, J. A. B., Murach, K. A., Dyar, K. A. \u0026amp; Zierath, J. R. Exercise metabolism and adaptation in skeletal muscle. \u003cem\u003eNat. Rev. Mol. Cell. Biol.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 607\u0026ndash;632 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoa Sorte Silva, N. C., Barha, C. K., Erickson, K. I., Kramer, A. F. \u0026amp; Liu-Ambrose, T. Physical exercise, cognition, and brain health in aging. \u003cem\u003eTrends Neurosci.\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 402\u0026ndash;417 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Fernandes, S. et al. M. S. 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Endocrinol.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 97\u0026ndash;108 (1976).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"serotonin, dopamine, single neurons, exercise, locomotion, adaptation","lastPublishedDoi":"10.21203/rs.3.rs-9290981/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9290981/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhysical exercise influences monoaminergic and neurotrophic systems in the brains of mammals and some invertebrates. Little is known about how a specific neuron adapts its biophysical properties to acute or repeated physical activity. Here we tested the hypothesis that central monoaminergic neurons develop a state of readiness for exercise by changing their baseline biophysical properties during regular training. In the mollusc Lymnaea stagnalis, we compared the activity of central monoaminergic neurons and brain monoamine levels immediately after a single two-hour exercise (crawling in low water) and one day after completion of the two-week daily two-hour exercise. Under both acute and baseline chronic conditions, serotonergic neurons that control locomotion were depolarized both in situ and being completely isolated. The dopaminergic neuron that controls respiration was hyperpolarized, and dopamine levels in the brain were reduced under both acute and chronic conditions, while a two-hour rest after acute exercise abolished these effects. Monoamine-dependent behaviors were also altered by regular exercise: the speed of locomotion, consumption and oviposition increased, while growth rate decreased. Our findings suggest that regular exercise alters the baseline biophysical properties of central monoaminergic neurons, potentially through an anticipatory mechanism preparing the nervous system for subsequent exercise.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Chronic exercise changes the baseline properties of central monoaminergic systems, identified neurons and related behaviors","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 13:32:11","doi":"10.21203/rs.3.rs-9290981/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-12T10:32:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T17:11:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-27T16:11:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"10383910936784875045840511615697431843","date":"2026-04-27T00:32:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"138423859997996877058931729245289037657","date":"2026-04-23T06:28:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123574189104745723578569537278153149440","date":"2026-04-20T11:39:03+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T10:42:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T07:37:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-06T14:59:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-04-06T13:35:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"98e12c90-57c3-4926-91d1-2b691d4bd14f","owner":[],"postedDate":"April 28th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-12T10:32:35+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-03T17:11:47+00:00","index":39,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":67107873,"name":"Biological sciences/Neuroscience"},{"id":67107876,"name":"Biological sciences/Physiology"}],"tags":[],"updatedAt":"2026-05-12T11:05:15+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 13:32:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9290981","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9290981","identity":"rs-9290981","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}