The prevailing orthodoxy is that single cells cannot learn; Beatrice Gelber & more recent studies suggest that such learning may be evolutionarily more widespread & fundamental to life than previously thought

Reconsidering the evidence for learning in single cells. Samuel J. Gershman et al. Dec 2020. https://gershmanlab.com/pubs/cell_learning.pdf

Abstract: The question of whether single cells can learn led to much debate in the early 20th century. The view prevailed that they were capable of non-associative learning but not of associative learning, such as Pavlovian conditioning. Experiments indicating the contrary were considered either non-reproducible or subject to more acceptable interpretations. Recent developments suggest that the time is right to reconsider this consensus. We exhume the experiments of Beatrice Gelber on Pavlovian conditioning in the ciliate Paramecium aurelia, and suggest that criticisms of her findings can now be reinterpreted. Gelber was a remarkable scientist whose absence from the historical record testifies to the prevailing orthodoxy that single cells cannot learn. Her work, and more recent studies, suggest that such learning may be evolutionarily more widespread and fundamental to life than previously thought and we discuss the implications for different aspects of biology.

Implications for the neurobiology of learning and memory

If single cells can learn, then they must be using a non-synaptic form of memory storage. The idea that intracellular molecules store memories has a long history, mainly in the study of multicellular organisms. We’ve already mentioned McConnell’s studies of planarians; similar ideas were espoused by Georges Ungar based on his studies of rodents (Ungar et al., 1968, Ungar and Irwin, 1967). These studies indicated that memories could be transferred from one organism to another by injection or ingestion of processed brain material. Clearly no synaptic information could survive such processing, so transfer could presumably only occur if the memory substrate was molecular. However, these findings were the subject of much controversy. The failure of careful attempts to replicate them led to a strong consensus against their validity and this line of research eventually died out (Byrne et al., 1966, Setlow, 1997, Smith, 1974, Travis, 1980). Nonetheless, several lines of recent work have revisited these studies (Shomrat and Levin, 2013, Smalheiser et al., 2001). For example, Bed´ ecarrats et al. (2018) showed that long-term sensitization of the siphon-withdrawal reflex ´ in Aplysia could be transferred by injection of RNA from a trained animal into an untrained animal. This study further showed that this form of transfer was mediated by increased excitability of sensory (but not motor) neurons, and depended on DNA methylation, although the study did not establish either RNA or DNA methylation as the engram storage mechanism. In another line of work, Dias and Ressler (2014) showed that fear conditioning in rodents could be transferred from parents to offspring, an effect that was associated with changes in DNA methylation. These studies not only revive the molecular memory hypothesis, but also point towards specific intracellular mechanisms. The significance of DNA methylation lies in the fact that DNA methylation state can control transcription. Thus, the set of proteins expressed in a cell can be altered by changes in DNA methylation, which are known to occur in an experience-dependent manner. For example, after fear conditioning, the methylation states of 9.2% of genes in the hippocampus of rats were found to be altered (Duke et al., 2017). As first pointed out by Crick (1984), and later elaborated by Holliday (1999), DNA methylation is a potentially stable medium for heritable memory storage, because the methylation state will persist in the face of DNA replication, thanks to the semi-conservative action of DNA methyltransferases. A related idea, put forward independently in Lisman et al. (2018), is that a stable memory could arise from the tug-of-war between enzymatic phosphorylation and dephosphorylation. In essence, the idea is to achieve stability through change: a molecular substrate maintains its activation state by means of continual enzymatic activity. Crick and Lisman suggested that this could solve the problem of molecular turnover that vexes synaptic theories of memory. Consistent with this hypothesis, inhibition of DNA methyltransferase disrupts the formation and maintenance of memory, although it remains to be seen whether methylation states themselves constitute the engram (Miller et al., 2010, Miller and Sweatt, 2007). The proposals of Crick and Lisman apply generally to enzymatic modification processes (e.g., acetylation or glycosylation) acting on macromolecules, provided 10 that the biochemical dynamics can generate the appropriate stable states (Prabakaran et al., 2012). An important distinction between the forms of dynamical information storage proposed by Crick and Lisman and the storage provided by DNA is that the latter is largely stable in the absence of enzymatic activity, under conditions of thermodynamic equilibrium. In contrast, the former typically relies on enzymatic activity and is only stable if driven away from thermodynamic equilibrium by chemical potential differences generated by core metabolic processes. In other words, the latter may accurately retain information in the absence of a cell over a substantially longer period than the former, which may lose information rapidly in the absence of supporting enzymatic activity. Another candidate medium for intracellular memory storage is histone modification. In eukaryotes, DNA is wrapped around nucleosomes, composed of histone proteins, to form chromatin. Gene transcription can be controlled by changes in the modification state (acetylation, methylation, ubiquitination, etc.) of these histones. In the cell biology literature, an influential hypothesis posits the existence of a histone “code” (Jenuwein and Allis, 2001, Turner, 2002) or “language” (Lee et al., 2010) that stores information non-genetically, although the nature of that information has been a matter of debate (Henikoff and Shilatifard, 2011, Sims and Reinberg, 2008). Early work demonstrated that learning was accompanied by increased histone acetylation in the rat hippocampus (Schmitt and Matthies, 1979), and more recent work has established that memory can be enhanced by increases in histone acetylation (Levenson et al., 2004, Stefanko et al., 2009, Vecsey et al., 2007). Bronfman et al. (2016) provide an extensive survey of the molecular correlates of learning and memory. In parallel with these findings, molecular biologists grappling with the information processing that takes place within the organism have begun to suggest that signaling networks may implement forms of learning (Csermely et al., 2020, Koseska and Bastiaens, 2017). In this respect, Koshland’s studies of habituation of signaling responses in PC12 cells, a mammalian cell line of neuroendocrine origin, are especially resonant (McFadden and Koshland, 1990). Koshland’s work was undertaken in full awareness of learning studies conducted by Kandel and Thompson in invertebrate organisms but his pioneering efforts have not been explored further. This reflects, perhaps, the intellectual distance between cognitive science and molecular biology, which the present paper seeks to bridge. The information processing demands on a single-celled organism, which must fend for itself, are presumably quite different from those confronting a single cell within a multi-cellular organism during development and homeostasis, so what role learning plays within the organism remains a tantalizing open question. 

Beatrice Gelber, though she could not have known about the specifics of DNA methylation or histone modification, was uncannily prophetic about these developments: 

This paper presents a new approach to behavioral problems which might be called molecular biopsychology... Simply stated, it is hypothesized that the memory engram must be coded in macromolecules... As the geneticist studies the inherited characteristics of an organism the psychologist studies the modification of this inherited matrix by interaction with the environment. Possibly the biochemical and cellular physiological processes which encode new responses are continuous throughout the phyla (as genetic codes are) and therefore would be reasonably similar for a protozoan and a mammal. (Gelber, 1962a, p. 166)

The idea that intracellular mechanisms of memory storage might be conserved across phyla is tantalizing yet untested. The demise of behavioral studies in Paramecia and other ciliates has meant that, despite the wealth of knowledge about ciliate biology, we still know quite little about the molecular mechanisms underlying Gelber’s findings. Nonetheless, we do know that many intracellular pathways that have been implicated in multicellular memory formation exist in ciliates (Table 1). For example, ciliates express calmodulin, MAP kinases, voltage-gated calcium channels, in addition to utilizing various epigenetic mechanisms that might be plausible memory substrates, such as DNA methylation and histone modification. In like manner, key molecular components of neurons and synapses emerged in organisms without nervous systems, including unicellular organisms (Arendt, 2020, Ryan and Grant, 2009). We believe it is an ideal time to revisit the phylogenetic origins of learning experimentally and theoretically