Evidence of conditioned behavior in amoebae

Figure 8a shows the galvanotactic locomotion of 50 cellular trajectories (experimental replicates: 4, number of cells per replicate: 4–15) analyzed under an external, controlled direct-current electric field of about 340 mV/mm. This study indicated that practically all the metamoebae migrated towards the cathode during 30 min. The quantitative analysis showed that the values of the cosines of displacements were distributed between 0.04 and 1 (0.98/0.09 median/IQ), which verified that a unique fundamental behavior characterized by an unequivocal directionality towards the cathode had emerged in the experimental system.

Next, 160 metamoebae (experimental replicates: 15, number of cells per replicate: 3–12) were subjected to an induction process of 30 min. They were exposed simultaneously to chemotactic and galvanotactic stimuli, placing the peptide on the anode side (Fig. 9). Under these conditions, the record of the migration trajectories showed that 39% of the metamoebae ignored the electric field signal and moved towards the anode, while the remaining (61%) migrated to the cathode. The cosines of the displacement angles were distributed between −1 and 1 (0.54/1.8 median/IQ). Hence, the result of the induction process indicated that two fundamental cellular migratory behaviors had emerged in the experimental system, one towards the anode and another towards the cathode. The Wilcoxon rank-sum test confirmed the presence of these two different behaviors (p = 10−26; Z = −10.639, Wilcoxon rank-sum test).

Finally, to verify whether the cells that moved towards the anode during the induction process presented some kind of conditioning in their migratory trajectories, we performed a conditioned behavior test (Fig. 8b). For such a purpose, the 62 Metamoeba leningradensis (experimental replicates: 15, number of cells per replicate: 1–7) that had previously migrated towards the anode-peptide during the induction process were manually extracted and placed for 5 min into a normal culture medium (Chalkley´s medium) in a small Petri dish, in absence of stimuli. Then, in a similar way that we did in the Amoeba proteus experiments, the metamoebae were placed usually in groups of 1–6 on a new identical glass and block set-up that had never been in contact with the chemotactic peptide. Under these conditions, they were exposed for the second time to a single electric field, without peptide, during 30 min (the total time after the induction process was 35 min).

The analysis of the individual trajectories showed that 71% of the cells ran to the anode where the peptide was absent (Fig. 8b). The cosines of displacements ranged between −1 and 0.99 (−0.539/1.33 median/IQ). This result supported mathematically that the majority of cells moved towards the anode in the absence of peptide, thus corroborating that a new locomotion pattern had appeared in the metamoebae cells. Such systemic behavior (migration towards the anode in the absence of peptide) had never appeared before. The comparison between the cosines of displacements obtained during the galvanotaxis without previous induction (Fig. 8a) and the galvanotaxis after the induction (Fig. 8b) showed that this newly acquired cellular behavior is extremely unlikely to be obtained by chance (p = 10−17; Z = 8.326, Wilcoxon rank-sum test) in Metamoeba leningradensis.

Here, using an appropriate direct-current electric field (galvanotaxis) and a specific peptide (nFMLP) as a chemoattractant (chemotaxis) we have addressed essential aspects of the Amoeba proteus and Metamoeba leningradensis migration. More precisely, we have found that these cells can link two different past events, shaping an associative conditioning process characterized by the emergence of a new type of systemic motility pattern. This behavior consists in a persistent migration towards the anode when these cells typically migrate to the cathode.

First we have studied the Amoeba proteus migration and we have verified in the galvanotactic experiments that practically all the amoebae show an unequivocal systemic response consisting in the migration towards the cathode when they are exposed to a strong direct electric field of about 300–600 mV/mm (Fig. 3b, and Fig. 6). However, if the amoebae are exposed simultaneously to a chemotactic and galvanotactic stimulus (induction process), placing a specific peptide in the anode, 53% of the amoebae moved to the site where the peptide was located, and most of these cells (82%) were able to acquire a new singular behavior in their systemic locomotion characterized by a persistent migration towards the anode, which was observed in subsequent galvanotaxis experiments carried out in absence of peptide (Fig. 4b–d).

This extensive study, which has covered 615 cellular trajectories, has shown that when the exposition to a stimulus related to the amoeba’s nourishment (a specific peptide) is accompanied by an electric field, and the peptide is placed in the anode, the amoebae appear to associate the anode with the food (the peptide) and after the induction process most cells developed a new persistent pattern of cellular motility characterized by movements towards the anode even if the nourishment (peptide) was absent. After an induction process, most of amoebae seem to associate food with the anode and, consequently, modify their conduct, behaving against their known tendency to move to the cathode. Strikingly, this induced association of anode and food can be maintained for relatively long periods of time. In our experiments, this conditioned motility pattern prevailed for periods ranging from 20 to 95 min. This period of time is very long in comparative terms if we take into consideration that the cellular cycle of Amoeba proteus lasts, with small variations depending on the environment, only about 24 h under controlled culture conditions22. We have also observed that, after the induction process, a small subset of the amoebae was not conditioned. Cells display a range of differences in their membrane receptors, electric potential, physiological/metabolic functioning, and hence, there are no two identical unicellular organisms. In our experiments, some cells probably were unconditioned or weakly conditioned due to their intrinsic physiological peculiarities, and in addition, some kind of cellular damage caused by the experimental process may have occurred.

To test the robustness of the conditioned behavior we have performed a preliminary study in Metamoeba leningradensis under the same strict conditions that we set up for Amoeba proteus. Despite these restrictive conditions, most metamoeba cells were able to link two different past events, same as Amoeba proteus, shaping an associative conditioning process characterized by the emergence of a new type of systemic motility pattern which consists in a persistent migration towards the anode when, in the absence of previous induction, these cells also typically migrate to the cathode (Fig. 8).

The controls carried out during the research indicated that cells exposed independently either to galvanotaxis or chemotaxis, did not present any observable atypical behavior (Fig. 6), and the quantitative study performed emphasized that it is extremely unlikely to obtain the new type of induced systemic behavior by chance (p = 10−19; Z = 8.878, Wilcoxon rank-sum test). In conclusion, the work we have performed here shows that most of the conditioned Amoeba proteus and Metamoeba leningradensis exhibited the ability to preserve the relationship between the two stimuli, acquiring a new type of systemic behavior via conditioning. Noteworthy, the fact that individual cells are able to generate associative conditioned behaviors to guide their complex migration movements has never been verified so far.

Our experimental results may allow another possible explanation. The exposure to nFMLP triggers a sub-population of cells to change the character of their migration in an electric field, making them to migrate towards the anode rather than the cathode. A notable number of cells belonging to both species can persistently change their migration pattern by these two external, simple and independent stimuli, when both are simultaneously applied. The new behavior persists for around 1 h, and gradually fades away thereafter.

Amoebae and metamoebae cells seem to associate the anode with the peptide in the induction process. After the conditioning, both stimuli seem to remain linked in these cells for a relatively long period of time, and consequently, the systemic movement of amoebae and metamoebae responded to the presence of an electric field by migrating towards the anode instead of the expected migration to the cathode.

In brief, we have observed a systemic cell behavior that can be modified by two simple external and independent stimuli, when they are simultaneously applied. This conditioned migration behavior can prevail for 44 min on average.

Pavlov studied four fundamental types of persistent behavior provoked by two stimuli. Here we have based our work in one of them, the called “simultaneous conditioning”, in which both stimuli are applied at the same time.

However, in a strict sense, we cannot conclude that our findings represent the classical Pavlovian conditioning since complete controls and parametric analyses for classical conditioning studies have not been performed yet23.

The experiments we show here were inspired by numeric predictions based on computational modeling that we published in 2013 dealing with complex metabolic networks24. Thus, analyzing complex enzymatic processes under systemic conditions using Statistical Mechanic tools and advanced Computational and Artificial Intelligence techniques, we were able to verify numerically that self-organized enzymatic activities in modular metabolic networks seem to be governed by Hopfield-like attractor dynamics similar to what happens in neural networks24. A key attribute of the analyzed metabolic Hopfield-like dynamics is the presence of associative memory. This quantitative study showed that the associative memory in unicellular organisms is possible24,25. Such memory would be a manifestation of emergent properties underlying the complex dynamics of the systemic cellular metabolic networks.

It is still too early to delineate the molecular mechanisms supporting this cellular associative conditioning. However, there are evidences of a functional memory, which can be embedded in multiple stable molecular marks during epigenetic processes25. Likewise, long-term correlations (mimicking short-term memory in neuronal systems) have also been analyzed in experimental calcium-activated chloride fluxes in Xenopus laevis oocytes26. On the other hand, different studies have described several molecular processes in which both prokaryotic and eukaryotic cells show chemotactic memory. For instance, changing dynamics in specific methylation-demethylation patterns in prokaryotes seem to be involved in molecular memory processes related to chemical gradient adaptation27–30. Besides, phosphotransfer processes and other post-translational modifications seem to be involved in chemostatic cellular persistence of eukaryotic cells31–33.

In this paper, we have addressed essential aspects of the Amoeba proteus and Metamoeba leningradensis migration. The mechanisms underlying amoeba locomotion are extremely complex and the ability to direct their movement and growth in response to external stimuli is of critical significance for its functionality; in fact, cellular life would be impossible without regulated motility. Although some progress is being made in the understanding of cellular locomotion, how cells move efficiently through diverse environments, and migrate in the presence of complex cues, is an important unresolved issue in contemporary biology. Free cells need to regulate their locomotion movements in order to accomplish critical activities like locating food and avoiding predators or adverse conditions. In the same way, cellular migration is required in multicellular organisms for a plethora of fundamental physiological processes such as embryogenesis, organogenesis and immune responses. In fact, deregulated human cellular migration is involved in important diseases such as immunodeficiencies and cancer11,34. Neoplastic progression (invasion and metastases), for example, can be regarded as a process in which the survival of tumor cells depends also on their ability to migrate to obtain additional resources in a general context of scarcity35.

Here, we have verified that two unicellular organisms such as Amoeba proteus and Metamoeba leningradensis are able to modify their systemic response to a determined external stimulus exclusively by associative conditioning. This fact opens up a new framework in the understanding of the mechanisms that underlie the complex systemic behavior involved in cellular migration and in the adaptive capacity of cells to the external medium.

Amoeba proteus (Carolina Biological Supply Company, Burlington, NC.Item # 131306) were grown at 21 °C on Simplified Chalkley’s Medium (NaCl, 1.4 mM; KCl, 0.026 mM; CaCl2, 0.01 mM), alongside Chilomonas as food organisms (Carolina Biological Supply Company Item #131734) and baked wheat corns.

Metamoeba leningradensis (Culture Collection of Algae and Protozoa, Oban, Scotland, UK, CCAP catalog number 1503/6). They were cultured in the same conditions as Amoeba proteus.

All the experiments were performed in a specific set-up (Fig. 1) consisting in two standard electrophoresis blocks, 17.5 cm long (Biorad Mini-Sub cell GT), a power supply (Biorad powerbank s2000), two agar bridges (2% agar in 0.5 N KCl, 10–12 cm long) and a structure made from a standard glass slide and covers commonly used in Cytology Laboratories.

The first electrophoresis block was directly plugged into the power supply while the other was connected to the first via the two agar bridges, which allowed the current to pass through and prevented the direct contact between the anode and cathode and the medium where the cells would be placed later. Both electrophoresis blocks consisted of 3 parts: on the extremes, there are 2 wells which were filled by the conductive medium (Chalkley’s Simplified Medium) and, in the middle, an elevated platform (Fig. 1).

In the center of the second electrophoresis block we placed the experimental chamber that allowed us to obtain a laminar flux when it was closed and the addition and extraction of cells when it was open.

The experimental chamber consisted in 4 pieces of glass (standard glass slide and covers commonly used in Cytology Laboratories), a 75 × 25 mm modified slide and three small pieces obtained by trimming of three cover glass of 60 × 24 × 0.1 mm (Fig. 1).

Three cover glasses were trimmed with a methacrylate ruler, one measuring about 3 × 24 × 0.1 mm and two measuring about 40 × 24 × 0.1 mm each, here on called central piece and sliding lateral pieces, respectively. These three glasses were for only one use.

This glass structure (Fig. 1) supported the sliding parts of the experimental chamber. It was reusable after cleaning. To build it, we fixed with silicone on a glass slide along the two longest sides of the slide (if the width of a cover is 24 mm, about 4 mm were stuck on the slide and 20 mm protrude towards the outside of the glass slide). Then we left it to dry for 24 h. The last step consisted on trimming the protruding portions of the cover slides (about 60 × 20 × 0.1 mm) with a methacrylate ruler, so that two small longitudinal strips of approximately 60 × 4 × 0.1 mm were adhered to the glass slide (see Fig. 1), which shaped the lateral limits of the experimentation chamber.

The modified slide was placed in the central platform of the second electrophoresis block. To avoid medium going across the modified slide from below, we placed an oil drop in the central platform of the block of electrophoresis, on which the modified slide was placed. It is very important that the oil drop expands to cover the entire width of the experimental chamber. In the center of the modified slide, without any glue, we placed the central piece and the two sliding lateral pieces leaving short distance between all of them.

The amoebae were placed below the central piece of the chamber in an approximate volume of 30 µl. To note, it is crucial that the amoebae do not remain for more than a few seconds in the micropipette tip to avoid the adhesion of the amoeba to the inner surface of the tip.

Once the amoebae were placed under the central piece of the chamber, we waited for two minutes to allow the cells to stick to the surface of the modified slide. Then, we filled the wells of the electrophoresis blocks with simplified Chalkley medium up to the level necessary to contact with the base of the modified slide, but not the two sliding lateral pieces. Later, the two sliding lateral pieces were pushed with two micropipette tips until they contact with the liquid in the wells. Next, the two sliding lateral pieces are pushed to contact the central piece in the chamber. This way, a laminar flux can be established throughout the inner space of the experimental chamber.

In the induction process, once the laminar flux was created, and before the activation of the electric field, we added 750 µl of 2 × 10−4 M nFMLP peptide solution to the medium (75 ml) in the positive pole well of the second electrophoresis block.