The Bioelectrics of Immortality and Mortality in Cold-Sensitive <i>Hydra oligactis</i>

This study provides a consensus whole-body bioelectric atlas of living immortal and mortal hydra. We confirmed that hydra were aging at 10°C since they had formed ovaries/testes within 78 days, an indication that this strain is undergoing irreversible aging.70,82

Our results showed that in this model system, the FluoVolt and VF2.1.Cl dyes were showing Vmem of the plasma membrane and not artifacts, as revealed by the lack of signal from a closely related control dye that is not responsive to membrane potential. This control has been used in cultures of rat hippocampal neurons, human cardiomyocytes, human embryonic kidney cells,83–85 and the tails of living frog embryos,68 but is not common and should be universally used in voltage dye studies.

Prior efforts have mapped action potentials of the anterior region of the guinea pig’s heart86,87; resting potential patterns in bacterial biofilms88; central regions of poplar leaves89 and stem and leaf of sunflowers Helianthus annuus90; the optic tectum region of the frog brain91; the olfactory bulb of the salamander Ambystoma tigrinum92,93; faces, brains, and tails of frog embryos30,68,72; olfactory bulb of zebrafish94,95; olfactory bulb of box turtles Terrapene triunguis96; an embryonic precontractile chick heart97; the pig heart98; mouse heart87,99; and the primary somatosensory cortex of the mouse brain.100 We show for the first time the relative membrane voltage of living cells across the whole body of 38 whole organisms. Certain properties of hydra and technological advances have made this possible. Specifically, hydra has a very thin layer of cells, and thus Vmem-sensitive dyes can easily penetrate these cell layers and be observed under the confocal microscope using FLIM that shows the relative membrane voltage of each cell.

A limitation to note in our data is that we cannot rule out changes in membrane voltage of cells that change their membrane voltage faster (within approximately a few milliseconds101) than the completion of the whole-body imaging of a hydra (a few hundred milliseconds: see the section “Methods”). Such signals are unlikely to be critical for developmental bioelectricity because the transduction machinery – and downstream effects on transcriptional changes, epigenetic processes, and cell behavior – all typically take minutes to hours to take place.102

With any dye, it is important to consider potential toxicity. We do not believe this was a factor in our study. Regarding potential dye toxicity, any dye toxicity would be present in both hydra conditions, and in the old mortal versus immortal hydra analyses, we are reporting differences in the relative bioelectric pattern of these two conditions that have both been exposed to the dye and washed using the same protocol. The hydra were alive after the brief exposure to the dye, and when they were imaged under the confocal. The laser line used to image the hydra was in the green region (510 nm) and thus relatively less cytotoxic than if a laser line in the blue region was used.68 If the dye was toxic, we would expect the entire body of the hydra to be depolarized; however, the hydra’s body was not entirely depolarized. Also, Miller et al. have found that VoltageFluor dyes have almost no capacitance compared with other Vmem sensors, so they have relatively little effect on Vmem.75 Additionally, exposure to the FluoVolt dye did not affect survival of the hydra or consistently change the morphology of the hydra 4 days after exposure to the dye (Supplementary Fig. S3 and Fig. S4)—a much longer time period than the minutes timescale on which we stained and imaged.

One of the key aspects of bioelectric patterns is that in some systems (Xenopus embryo, regenerating planaria, chick embryo), their distribution has been shown to serve as an instructive signal for morphogenesis.30,103–106 Thus, we were very interested in differences in Vmem across the hydra that would correspond to distinct anatomical features. We found that the foot of the hydra was more depolarized than other regions of the hydra’s body. The tentacles of the hydra were also relatively depolarized in some individuals. This relative depolarization may relate to the adhesive properties of the foot and secretory properties of the hydra’s tentacles. The hydra’s foot (also called basal disc) secretes granules that contain glycans and/or glycoproteins, allowing the hydra to stick on surfaces.107 Although the resting membrane potential of the secretory cells of the foot has not been previously reported, other secretory cells, called nematocytes, in the tentacles of Hydra vulgaris are known to require apical plasma membrane depolarization for the secretion of stenoteles.108

As with any biological parameter, we observed some variability across animals. The standard deviation in mean lifetime intensity was higher among the biological than among the technical replicates. Specifically, the standard deviation in mean lifetime intensity among the biological replicates of the data shown in Figure 3 and Figure 4A was 101 and 79 picoseconds in the case of the immortal hydra and old mortal hydra, respectively; whereas the standard deviation in mean lifetime intensity among the technical replicates (N = 2; Table 1) was 4–46 ps in the case of four immortal hydra and 4–47 ps in the case of four old mortal hydra. Although the standard deviation in mean lifetime intensity of the technical replicates was only measured from two technical replicates, the higher standard deviation in the case of the biological replicates compared to that of the technical replicates indicates that each hydra does not have the same whole-body mean lifetime intensity as other hydra. There tends to be more interindividual variation in the bioelectric pattern of hydra than temporal (1–3 min) variation in the bioelectric pattern of a single individual. In other words, the lower standard deviation in the case of the technical replicates, versus biological replicates, indicates that data within the ∼4–47 ps standard deviation could be considered temporal intraindividual variation (“noise”) and data beyond the ∼47 ps standard deviation could be considered interindividual variation (“signal”). Future work on the functional role of this parameter in hydra biology will be necessary because it is the precision of interpretation of the voltage profile by body systems that would determine the degree of precision that is physiologically important.

One basic bioelectric parameter to compare in living samples is simply the overall average level of polarization. With respect to this, we found a more hyperpolarized bioelectric pattern in the whole body of 2- to 4-month-old mortal than in immortal hydra. Τhe difference cannot be due to temperature per se since we collected all voltage data at the same temperature. What we observed may be due to dynamic changes among many different types of interacting cells. To be more specific, what we found was reasonable for two reasons. First, we would expect the immortal hydra’s foot and tentacles to be relatively depolarized, due to the depolarization required for excretion of substances in secretory cells present in those regions. If this depolarization becomes dysregulated during aging, we would expect old hydra to be, on average, more hyperpolarized than immortal hydra. Second, hyperpolarization of the plasma membrane is known to determine actin fiber compaction in cultures of bovine epithelial cells,109 and actin fibers appear disrupted in aging hydra.71 Thus, although these two processes happen in very evolutionary distant species, the disrupted actin fibers of aging hydra may be due to cell hyperpolarization.

There seems to be some variety across organisms in terms of the relationship of polarization level to aging. For example, Type II neurons in the bed nucleus of the stria terminalis are more hyperpolarized in aged mice than in young mice.110 In rats, old versus young hippocampal CA1 neurons did not have significantly different resting membrane potentials.111 Also, there was no significant difference in the resting membrane potential of old versus young pyramidal neurons of the prefrontal cortex of rhesus monkeys.112 In female and male mouse bladder cells, the messenger RNA expression of the hcn1 gene, which encodes the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, decreases with age.113 H. vulgaris also has HCN channels (https://www.ncbi.nlm.nih.gov/gene/100208250; https://www.ncbi.nlm.nih.gov/gene/105844959) which are approximately 1 substitution per site, on the protein sequence, different than the human HCN1-4 channels.114 If hnc1 expression is lower and production of HCN1 proteins is decreased in aging hydra, this may explain the potential inability of cells to maintain their depolarized state as they age, that is, our finding that older mortal hydra are more hyperpolarized than immortal hydra.

In addition to average polarization, it is interesting to consider the crispness of the bioelectric pattern itself: the degree to which bioelectric patterns sharply distinguish compartments with different Vmem.22,115 In embryos, a number of teratogens that disrupt developmental morphogenesis do so by making the bioelectric prepattern less sharp.72,116 For example, in the Xenopus brain, this is known to be the case for chemical and genetic teratogens; crucially, brain morphogenesis, gene expression, and even learning rates can be restored despite exposure to nicotine, ethanol, or mutation of the Notch gene by artificially forcing the bioelectric pattern to become sharper (via overexpression of hcn2, which encodes an ion channel).117–119 We previously suggested the hypothesis that aging could involve a change of the sharpness of the bioelectrical pattern25 and indeed hcn1 expression decreases with age in mice.113 We thus quantified here the sharpness of the bioelectrical patterns in immortal and aging hydra. We found a sharper bioelectric pattern in the whole body of old mortal than immortal hydra. However, the foot-to-central body ratio pattern was less sharp in the old mortal hydra. This suggests either that different organisms handle this in different ways, or more likely, that the relationship between crispness of Vmem pattern and aging is not uniform across body regions.

Our work points to the need for numerous subsequent studies. In parallel with applying antiaging bioelectric treatments (e.g., hcn2 overexpression) in hydra, it would be useful to understand the mechanisms underpinning the observed whole-body bioelectric atlas of hydra. What ion channels and pumps determine the membrane voltage of every cell? Are there particular groups of cells that have more coordinated/synchronized changes in membrane voltage than other more distant groups of cells in the hydra’s body? How exactly are the known aging-related physiologies of hydra (decreased ability to capture prey, decreased ability to spontaneously contract and transfer food to the gut, fewer sperm and nurse cells after 2 months, fewer interstitial stem cells, and fewer epithelial cells71) associated with the bioelectric atlas of old mortal hydra? Relative hyperpolarization of the old hydra’s body may also mean a hyperpolarization of the depolarization-activated nematocytes in the hydra’s tentacles, thus contributing to their decreased ability to capture food. However, the associations between the other old physiologies and the old mortal bioelectric atlas are largely unknown. The availability of protocols for generating transgenic hydra120–123 suggests the possibility of using optogenetics and ion channel misexpression to understand the role of bioelectrics in hydra morphogenesis and aging. Likewise, it would be interesting to understand the role, if any, of endogenous bioelectrical states and the abilities of external alternating electric fields to induce reversal and continuation of hydra morphogenesis.124 Finally, since applied electric fields have been shown to induce reversible changes in hydra morphogenesis,124,125 the future study of bioelectrics in various aspects of hydra biology is likely to reveal much of interest.

Identification of the whole-body bioelectric atlas of immortal and aging mortal hydra is a step toward performing similar studies in other model and nonmodel organisms. Studying the bioelectrics of aging in different taxa across the tree of life and the evolution of bioelectric pathways will help better understand the bioelectrics of aging in clinical settings. Examining the bioelectrics of human cells and tissues in both young and elderly populations would be essential in designing best practices for early human clinical trials using morphoceuticals25 or gero-electroceuticals,38 that is, drugs that target cellular bioelectrics and aim to reverse aging and aging-related diseases. Comparative developmental electrophysiology across widely distributed phyla is a first step in cracking the bioelectric code toward a better understanding of evolutionary physiology and the biomedicine of aging and disease.

We thank the Brigitte Galliot’s Lab for providing the CS H. oligactis; Erin Switzer for providing the artemia brine shrimps; Hamid Sediqi for sharing the code for calculating Moran’s I; Aviva Lucas-Demott and Alexandra Griffin for helping maintain the hydra; and Julia Poirier for assistance with the article.

S.E.K.: Conceptualization, data curation, supervision, software, formal analysis, validation, visualization, investigation, methodology, project administration, writing—original draft, and writing—review and editing. A.P.: Data curation and investigation. P.M.: Supervision, software, project administration, data curation, resources, visualization, investigation, methodology, and writing—review and editing. P.T. and P.E.: Supervision. M.L.: Conceptualization, supervision, resources, funding acquisition, project administration, visualization, methodology, and writing—review and editing.

M.L.’s lab has a sponsored research agreement with Astonishing Labs, a company seeking to advance the biomedicine of aging. All the other authors have no conflicts of interest to disclose.

This research was supported by a sponsored research agreement from Astonishing Labs.