动物实验 z-bio 2021-10-19 365

　　Introduction: In the whole vertebrates, the ion concentration in plasma remains basically unchanged, sodium ions and chloride ions are present above 100 mM, and all other major ions remain below 5 to 10 mM. However, the mechanisms used by animals for osmotic regulation are diverse and depend on their environment. Land animals and marine mammals balance ion-rich foods or liquids by ingesting ion-poor water and excreting excess ions. On the other hand, fish and amphibians supplement water consumption and excretion through a special system of epidermal cells and ion cells, which can exchange ions with the surrounding water. Importantly, the direction and mechanism of ion exchange depend on the salinity of the animal's natural environment. The ion cells of freshwater fish must absorb salt, while the ion cells of sea fish discharge salt. Terrestrial animals contact and balance salt through food intake, leading to the evolution of their taste system to be highly sensitive to salt. They usually use two separate channels to distinguish appetite (<100>150 mM) NaCl concentration. In vertebrates, they are respectively mediated by epithelial sodium channels, which specifically allow sodium influx, while bitter taste receptors are widely controlled by harmful stimulants. In contrast, fish’s main external salt sensors are unknown at the tissue and molecular levels. Aquatic animals are directly exposed to changes in environmental salinity, which suggests that it may be advantageous to combine their salt detection with various methods suitable for environmental navigation, such as smell, somatosensory, separate chemical sensors on the body and gills . Although the taste system of the bony fish is obviously related to feeding-related behaviors, it is unlikely to play a key role in the navigation of the salt gradient, because the bony fish has lost all homologues of the mammalian epithelial sodium channel family, and the bitter taste receptors are related to salt. No response to concentration. Therefore, it is not clear which molecular mechanism fish uses to detect external sodium chloride and adjust its behavior appropriately. Since the environment in which zebrafish may evolve is characterized by sharp changes in local salinity levels, the ability to detect and navigate the sodium chloride gradient will benefit them. This change can lead to increased stress and cortisol levels, which are ultimately fatal, making the neural mechanisms of detecting and avoiding salt gradients vital to survival. It shows that zebrafish has evolved a behavioral strategy to avoid high-salt environments, and this behavior is mediated by a subset of olfactory sensory neurons through the olfactory system, which can detect the presence of a combination of sodium and chlorine.

　　 Zebrafish larvae avoid high salt environment: In order to test whether zebrafish larvae can avoid high salt environment, we developed a method to observe the larvae swimming in the salt gradient in detail. In this experiment, four larvae were placed in separate lanes, fixed by two agar pads, and the two agar pads were made of filtered fish rearing water (control) or fish rearing water plus test salt (source). . It shows that the diffusion of sodium chloride (NaCl) from the source will produce a continuous salinity gradient across the entire lane (Figure S1A), and the larvae traveling in this gradient leave the source significantly longer than the control without salt. During the first 15 minutes, this disgust appeared along with the salt concentration gradient and stabilized thereafter. This behavior is powerful and indicates that the larvae 5 days after fertilization (dpf) have been equipped with aggressive strategies to avoid areas of high salinity. External sodium chloride fluctuations can change some environmental parameters, such as osmotic pressure, net conductivity, and specific ion characteristics. Any of these may be detected by the larvae and used to avoid high salt. In order to analyze the correlation of each parameter, we tested the larvae's preference for a series of compounds that separate specific factors. For example, the general aversion to the increase in osmotic pressure was tested by generating an equimolar sugar alcohol and mannitol gradient with the previously tested NaCl gradient. Under these conditions, the larvae have no position preference. In contrast, zebrafish reliably avoided every ionic solution we tested, whether it was composed of chloride and another monovalent (KCl) or divalent cation (MgCl2) or sodium. Compared to sodium chloride, larger ions (ie rubidium or bromide) are weaker and zebrafish larvae are easier to avoid, although this may be due to slower diffusion and lower concentration gradients. It is still unclear whether the mechanisms that avoid these other ions belong to the same or parallel neural pathways.

　　 Avoid salt by detecting increased salt: I hope to understand the specific heuristics used by zebrafish larvae to avoid high salt concentrations. We observed that, under all conditions, the larvae mainly swim back and forth on both ends of the arena, while lining up parallel to the longitudinal axis. However, when the larvae encounter a salt gradient rising, they will reverse direction earlier than when the gradient descends. This indicates a biased random walk, increasing the salt concentration amplifies the frequency of larval relocation. To test this hypothesis, we compared the distribution of redirection angles after swimming towards the salt and after swimming away from the salt. In the absence of gradients, the larvae in these two cases will not change their turn statistics. In contrast, in the gradient guidance process, if the larvae were brought closer to the salt rather than away from the salt in the previous round, the larvae are more likely to perform a 20 to 40 degree turn. Compared with the control condition, when the larvae encounters an ascending gradient, the steering amplitude is only adjusted by the salt concentration. The magnitude of this adjustment increases with the higher salt level in the source, indicating that the increase in salinity is not the absolute concentration driving the steering . In order to verify whether the biased random walk is sufficient to explain the avoidance behavior of the larvae, we use natural swimming and turning statistics to simulate the navigation ability of the virtual larva on the salt gradient. Turning is adjusted based on the absolute salinity level, or the probability of turning increases after a relative increase in salinity. We found that only the latter can fully capture the avoidance behavior of real animals. The underlying concern is that, according to the established model, the model predicts that the larvae will easily swim in water that is not salted at all. This is counter-intuitive because it will have obvious harmful effects on animals. By testing this prediction with deionized water, we found that the larvae did swim towards the lowest salt concentration, indicating that the larvae might not seek the best external NaCl setting, but always avoid increasing salinity. The possible explanation for this simple, even maladaptive strategy is that areas with extremely low salinity are very rare and may not exert any selective pressure on these animals. So far, we have confirmed that zebrafish larvae avoid high salinity waters and do so by responding to increased salt concentration. Next, we want to determine how to detect salt. In order to identify this sensory area, we hope to use calcium imaging technology to fix our animal's head. In order to adapt to this limitation, we designed a stimulation device that can quickly and reversibly present reversible chemicals on the face of the larva, while the head and trunk are embedded in agarose, and the tail can move freely. We found that the larvae's response to salt pulses is a strong tail flick and is concentration-dependent. Consistent with the free swimming behavior, the larvae reacted most sensitively at the beginning of the NaCl pulse, corresponding to the recent increase in concentration. Therefore, we consider this preparation as a reasonable substitute for larvae to swim freely in a concentration gradient under natural conditions. The activities of the olfactory and lateral line systems reflect external salinity: in order to screen the brain regions most sensitive to such NaCl pulses, we combined the restraint preparation with a customized light sheet microscope. This allows us to perform volumetric imaging across most of the brain in transgenic larvae expressing GCaMP6 under the control of the pan-neuronal HuC promoter, while delivering pulses of different NaCl concentrations and simultaneously tracking its behavior. After imaging, we use an algorithm based on time correlation to segment the fluorescence on each plane into active units. This algorithm only uses cross-time correlation and does not contain anatomical features, so these "active units" may consist of single cells, nerve fibers, or a combination of both. In order to identify any area carrying salinity information, we calculated the mutual information between each unit and the transported salt concentration. Taking the average of this value on each voxel of the fish's Z-brain map reveals the typical strong NaCl performance in the fish's olfactory system. In all sensory ganglia, only the most of the nerve fibers and olfactory epithelial cells at the tip contain units that reflect the concentration of NaCl. In addition, unlike the behavior that only subsides for a few seconds in the pulse, these areas maintain or even increase calcium levels throughout the stimulation period. This indicates that the olfactory epithelium and neuroticism of the lateral line contain sensory manifestations of external salt concentration, and cannot alone reflect when the animal responds. As predicted, the classifier can independently predict the stimulus, but it cannot predict whether the animal has a behavioral response.

　　 Olfactory input is necessary to drive NaCl avoidance behavior: In the next step, we want to clarify the exact role of the olfactory system and lateral lines in generating salt avoidance behavior. To this end, we cultivated larvae in different concentrations of copper sulfate, which can kill lateral line and olfactory epithelial cells. After the treatment and recovery period, we examined the behavioral response of the larvae to 50mM NaCl pulses and found that their response rate decreased with increasing copper concentration. In fact, when the copper sulfate concentration is 20 μM, the behavioral response to NaCl is basically eliminated. In order to ensure that copper does not simply reduce all motor abilities, we verified that copper-treated larvae did not reduce the performance of visual motor responses, an innate visual motor behavior. In order to distinguish the relative importance of smell and lateral lines, we checked whether the degree of damage in both ways can predict behavior. In order to quantify the remaining salt-induced calcium activity in these areas, we hope to obtain higher spatial resolution through light sheet microscopy; therefore, we performed two-photon imaging to evaluate the degree of sensitivity to NaCl removal after copper treatment. We found that treatment with copper sulfate as low as 2 μM had eliminated all lateral reactivity, even if the animal continued to respond to NaCl. In contrast, at this concentration, a large portion of NaCl-sensitive olfactory bulb units still remain responsive. Like this behavior, only 20 μM copper sulfate can completely eliminate these reactions, which means that the olfactory system is essential to avoid NaCl. In order to determine whether specific resection of nasal nerve hypertrophy can also reduce NaCl-triggered behavior, we performed targeted two-photon laser ablation of two nerve hypertrophy. We found that fish receiving this treatment or false ablation did not significantly reduce their behavioral response rate, indicating that the effect of these neurohypertrophy is minimal. To further verify the necessity of smell for NaCl-triggered behavior, we next performed a rough but informative experiment; we rotated the fish 180° relative to the stimulus. This allows us to expose the nerve endings and various other somatosensory systems in the tail to NaCl, while leaving the face and related olfactory systems unaffected. Fish are much less likely to respond, which supports that olfactory exposure is necessary to cause NaCl avoidance behavior. Neurons that are sensitive to NaCl are driven by sodium and chlorine: After confirming that sodium chloride does not directly depolarize all epithelial neurons, we next test whether the olfactory response is specifically driven by sodium and/or chloride . To this end, we provide a 50 mM NaCl random pulse for a given fish to identify NaCl sensitive areas or "test" chemicals. Reviewing the behavioral results in gradient analysis, we believe that NaCl-sensitive neurons are not osmotic pressure sensors. In fact, we found that NaCl-sensitive cells do not respond to equimolar mannitol. But the larva's avoidance of all ionic solutions did not provide clues about how NaCl-sensitive neurons might respond. We tested a series of solutions to analyze them. When we expose the larvae to KCl, KCl actually exchanges sodium for comparable monovalent ions, thereby strongly activating potassium, NaCl-sensitive neurons. Show that they are not sodium specific. To determine whether chloride drives this activity, we tested the combination of sodium with different anionic iodine. Overall, these results indicate that NaCl-sensitive cells have at least reported the presence of monovalent ions. However, we found that they are not conductivity sensors because when fish are exposed to isoelectric or higher concentrations of divalent cations (magnesium chloride), these cells respond significantly weaker than NaCl. Based on these results, we propose that the zebrafish's NaCl-sensitive neurons are mainly adapted to the presence of sodium and chlorine, and may be slightly more sensitive to sodium. To our knowledge, this dual sensitivity implies a new and undescribed molecular mechanism for environmental salt detection.