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date: 06 December 2019

Caenorhabditis elegans Learning and Memory

Summary and Keywords

The nematode, Caenorhabditis elegans (C. elegans), is an organism useful for the study of learning and memory at the molecular, cellular, neural circuitry, and behavioral levels. Its genetic tractability, transparency, connectome, and accessibility for in vivo cellular and molecular analyses are a few of the characteristics that make the organism such a powerful system for investigating mechanisms of learning and memory. It is able to learn and remember across many sensory modalities, including mechanosensation, chemosensation, thermosensation, oxygen sensing, and carbon dioxide sensing. C. elegans habituates to mechanosensory stimuli, and shows short-, intermediate-, and long-term memory, and context conditioning for mechanosensory habituation. The organism also displays chemotaxis to various chemicals, such as diacetyl and sodium chloride. This behavior is associated with several forms of learning, including state-dependent learning, classical conditioning, and aversive learning. C. elegans also shows thermotactic learning in which it learns to associate a particular temperature with the presence or absence of food. In addition, both oxygen preference and carbon dioxide avoidance in C. elegans can be altered by experience, indicating that they have memory for the oxygen or carbon dioxide environment they were reared in.

Many of the genes found to underlie learning and memory in C. elegans are homologous to genes involved in learning and memory in mammals; two examples are crh-1, which is the C. elegans homolog of the cAMP response element-binding protein (CREB), and glr-1, which encodes an AMPA glutamate receptor subunit. Both of these genes are involved in long-term memory for tap habituation, context conditioning in tap habituation, and chemosensory classical conditioning. C. elegans offers the advantage of having a very small nervous system (302 neurons), thus it is possible to understand what these conserved genes are doing at the level of single identified neurons. As many mechanisms of learning and memory in C. elegans appear to be similar in more complex organisms including humans, research with C. elegans aids our ever-growing understanding of the fundamental mechanisms of learning and memory across the animal kingdom.

Keywords: C. elegans, learning and memory, mechanosensory, chemosensory, thermosensory, habituation, context conditioning, state-dependent learning, classical conditioning, aversive learning, CREB, glutamate

Introduction

Caenorhabditis elegans (C. elegans), a 1-mm-long free-living, soil-dwelling nematode (roundworm), was chosen as an organism to study how genetics regulate development by Sydney Brenner in the 1960s (Brenner, 1973, 1988) because of its short life span (roughly 2–3 weeks) and rapid reproductive cycle (roughly 3 days), which allow large populations of animals to be rapidly grown in the laboratory. It quickly adapts to new environments and survives freezing, and is therefore easy to maintain in the laboratory.

Over the past few decades, an abundance of knowledge and resources about this powerful organism has been amassed. C. elegans was the first multicellular animal to have its genome fully sequenced (The C. elegans Sequencing Consortium, 1998). It contains approximately 20,000 genes, with approximately 38% of its genes having human homologs (Hodgkin, 2001; Shaye & Greenwald, 2011). The continually growing library of C. elegans mutant strains is enormous, as there are currently over 19,000 strains available from a central repository. Since C. elegans can survive extreme temperatures, these strains can be simply stored in liquid nitrogen or at −80°C. The entire cell lineage of every one of the 959 C. elegans cells from zygote to adult is known (Sulston, Schierenberg, White, & Thomson, 1983). It is currently the only multicellular organism to have its connectome completed (White, Southgate, Thomson, & Brenner, 1986).

As C. elegans is transparent, the organism is amenable to in vivo cellular and molecular analyses through various microscopy and light-based techniques, such as fluorescence imaging and optogenetics. For instance, genetically engineered proteins tagged with green fluorescent proteins (GFP) can be easily expressed in C. elegans (Hobert & Loria, 2006). Green fluorescent proteins have many uses, including visualizing cellular processes and determining gene expression patterns. Calcium imaging, which uses genetically encoded calcium reporters to monitor the calcium activity in neurons, has been extensively used in C. elegans, and allows for straightforward, economical in vivo imaging of neuronal activity Chung, Sun, & Gabel, 2013). In addition, optogenetics is a technique which uses proteins called opsins that respond to various wavelengths of light in order to activate or inhibit neurons (Deisseroth, 2010). For example, a neuron expressing a gene for an excitatory opsin (i.e., channelrhodopsin-2; ChR2) can be activated by blue light on the worm, while a neuron with halorhodopsin (NpHr) can be inhibited by yellow light. These are all powerful techniques that have been helpful in elucidating the mechanisms responsible for various types of behaviors in C. elegans.

Although C. elegans has only 302 neurons, this seemingly simple organism is able to learn and remember across many modalities. To date, many of the mechanisms underlying learning and memory in C. elegans appear to be fundamental, and conserved in more complex organisms, including humans.

Mechanosensory Learning and Memory

Introduction

Early research on Caenorhabditis elegan (C. elegans) showed that its development was determinate (Sulston et al., 1983), which would suggest that this animal was unable to learn and remember; however, this turned out not to be the case. The first type of learning discovered in C. elegans was habituation to a mechanosensory stimulus (Rankin, Beck, & Chiba, 1990). Habituation is a form of non-associative learning whereby an organism decreases its response to repeated sensory input (Rankin et al., 2009). In this paradigm, the worms received a mechanical stimulus (i.e., a tap), to the side of the agar-filled petri plate in which they resided. This tap resulted in the worms changing their behavior from crawling forward to crawling backward for a brief period of time—this reversal response is called the tap withdrawal response (TWR) (Bozorgmehr, Ardiel, McEwan, & Rankin, 2013). After repeated administrations of the tap, the worms responded less (i.e., they became habituated). When a noxious stimulus, such as a brief electric shock to the agar surface, was presented to habituated worms, dishabituation occurred—the worms showed an immediate significant increase in the response above the habituated level. Dishabituation is used to differentiate habituation from decrement resulting from sensory adaptation and/or fatigue.

The TWR of C. elegans varies with the worms’ age. When measuring the probability of responding (the number of worms responding to each tap of a habituation series), older worms habituated more deeply than younger worms (Timbers, Giles, Ardiel, Kerr, & Rankin, 2013). These age-related changes were hypothesized to be due to the aging worms’ declining ability to discriminate stimuli of different intensities. Because of these findings, tap habituation experiments are usually carried out with age-synchronized adult worms, 3–4 days post-hatch.

Caenorhabditis elegans Learning and MemoryClick to view larger

Figure 1. The tap withdrawal response circuit. All neurons are bilaterally symmetrical (left and right), except for AVM and DVA. The mechanosensory neurons are shown as blue ovals, the interneurons as yellow triangles, and the motor neurons as red rectangles. Chemical connections are represented by arrows, while electrical connections are represented by dashed lines. The thickness of the arrows and lines reflects the relative number of synapses. PVD and DVA neurons are shown as a green oval and triangle respectively, and they mediate both forward and backward movement. They may have a role in the integration of the two movements.

Adapted from Bozorgmehr et al. (2013).

The neural circuitry underlying the TWR was mapped using laser ablation experiments in which a laser was used to kill pairs of, or individual neurons (Chalfie, Sulston, White, Southgate, & Thomson, 1985; Wicks & Rankin, 1996) (Figure 1). The circuit consists of mechanosensory neurons, interneurons, and motor neurons. The mechanosensory neurons consist of two ALM neurons and a single AVM neuron in the anterior of the worm that sense head touch, while two PLM neurons in its posterior sense tail touch. The PVD neurons integrate sensory inputs coming from both the head and tail. Stimulating the anterior mechanosensory neurons causes a reversal, and stimulating the posterior mechanosensory neurons causes a forward acceleration (Bozorgmehr et al., 2013). The anterior and posterior mechanosensory neurons synapse onto the premotor interneurons, AVD, AVB, AVA, and PVC, which then synapse onto motor neurons that generate forward and backward movement. When all five mechanosensory neurons are simultaneously activated by a nonlocalized mechanical stimulus such as a tap, a reversal response is elicited—this is an integration of reversal and forward acceleration from the head and tail neurons (Bozorgmehr et al., 2013). The backward movement seen is hypothesized to occur because there are more anterior mechanosensory neurons than posterior ones (three anterior vs. two posterior), causing greater activation of the anterior touch circuit, which results in a reversal instead of a forward acceleration.

Studies have shown that this supposedly simple form of learning, habituation to tap, is comprised of several different behavioral parameters that are each regulated by different and interacting gene networks (McDiarmid, Yu, & Rankin, 2017).

Short-Term Memory (STM) for Tap Habituation

Caenorhabditis elegans can display short-, intermediate-, and long-term memory for tap habituation.

Short-term habituation is induced by delivering 30 or more taps at a 10 or 60-s interstimulus interval (ISI) (Rankin & Broster, 1992). Depending on the ISI, the depth and duration of short-term habituation varies: Habituation to stimuli presented at shorter ISIs leads to faster, deeper habituation and faster, more complete spontaneous recovery from habituation (Figure 2). This ISI-dependent behavior is similar to what is seen in habituation studies in other organisms, such as the gill-withdrawal reflex of Aplysia californica (Byrne, 1982). In addition, ISI-dependent recovery is another way of distinguishing habituation from sensory adaptation and fatigue.

Caenorhabditis elegans Learning and MemoryClick to view larger

Figure 2. Short-term habituation is different depending on the ISI presented. Compared to stimuli presented at a longer ISI (i.e., 60-s ISI), stimuli presented at a shorter ISI (i.e., 10-s ISI) lead to faster, deeper habituation.

Since glutamate neurotransmission was known to play an important role in learning and memory in mammals, the first candidate gene studied in tap habituation was eat-4, which encodes a glutamate vesicular transporter expressed in the mechanosensory neurons. EAT-4 is homologous to the mammalian vesicular glutamate transporter, VGLUT1. C elegans mutants, containing a loss-of-function eat-4 allele, are defective in glutamatergic neurotransmission (Lee, Sawin, Chalfie, Horvitz, & Avery, 1999). These eat-4 mutants showed a normal TWR, but showed faster and deeper habituation than wild-type worms at both a 10- and 60-s ISI (Rankin & Wicks, 2000). They also showed slower spontaneous recovery than wild-type worms. Interestingly, they failed to dishabituate after a brief electric shock. These results suggest that glutamate plays an important role in STM for tap habituation and also dishabituation.

Dopamine neurotransmission also modulates STM for tap habituation (Sanyal et al., 2004). The gene dop-1 encodes a D1-like dopamine receptor, which is expressed in the mechanosensory neurons (Sanyal et al., 2004). Compared to wild-type worms, dop-1 mutants showed a faster decline in the number of worms responding to repeated taps (i.e., reversal probability). Interestingly, dop-1 mutants displayed normal reversal distance during habituation compared to wild-type worms. Since mutations in dop-1 only altered reversal probability and not reversal distance, this was the first study suggesting that habituation of different components of the response (i.e., reversal probability and reversal distance) are mediated by different molecular mechanisms (Sanyal et al., 2004). In addition, another mutant strain deficient in dopamine neurotransmission, cat-2 (encodes tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis), showed a similar phenotype to dop-1 mutants that could be rescued by exogenous dopamine (Kindt et al., 2007). Interestingly, habituation to tap was only altered by dopamine if the worms were in the presence of bacteria (their food source).

Interestingly, there is in vivo genetic evidence that tap habituation can involve different mechanisms when stimuli are presented at different ISIs (Timbers et al., 2017). Two proteins, CMK-1 (ortholog of mammalian calcium/calmodulin-dependent kinases, CAMKI/IV) and OGT-1 (an O-linked β‎-N-acetylglucosamine transferase and a potential CAMK phosphorylation target), were found to differentially alter habituation at a 10 and 60 s ISI. Mutations in two genes, cmk-1 and ogt-1, affected the rate of response decrement at both a 10 and 60 s ISI and the final asymptotic level of habituation at a 60 s ISI, but not the final level at a 10 s ISI.

Long-Term Memory (LTM) for Tap Habituation

Caenorhabditis elegans (C. elegans) can form long-term memory (LTM) for tap habituation. When worms received habituation training 24 h or more before testing, they showed a smaller response to taps compared to worms which did not undergo habituation training and had only received a single tap stimulus (Rose, Kaun, Chen, & Rankin, 2003). The protocol for inducing LTM in C. elegans consisted of spaced/distributed training in which 4 blocks of 20 taps were delivered at a 60-s interstimulus interval (ISI) with a 1-h rest period between each block (Figure 3). When this protocol was carried out at a 10-s ISI, the worms did not show LTM. These results at a 10-s ISI and a 60-s support the hypothesis of multiple mechanisms of habituation. Similar to LTM in other organisms, LTM for tap habituation in C. elegans is dependent on protein synthesis, can last for more than 24 h, and is produced by spaced training (as opposed to massed training with no rest periods between training blocks).

Caenorhabditis elegans Learning and MemoryClick to view larger

Figure 3. The long-term memory (LTM) protocol involves trained worms receiving spaced training, where 4 blocks of 20 taps are delivered at a 60-s ISI with 1 rest period between each training block, while control worms receive 1 tap. After 24 h, 10 taps at a 60-s ISI are delivered to both control and trained worms to test for memory. The intermediate-term memory (ITM) protocol involves trained worms receiving massed training with 80 taps at a 60-s ISI, while control worms receive 1 tap. After 12 h, 10 taps at a 60-s ISI are delivered to both control and trained worms to test for memory.

The cAMP response element-binding protein (CREB) is a transcription factor that mediates protein synthesis that is important for long-term memory in most species. C. elegans, with a loss-of-function mutation in CRH-1, the C. elegans homolog of CREB, did not exhibit LTM for tap habituation (Timbers & Rankin, 2011). The LTM deficiency of crh-1 mutants was rescued by expressing wild-type CRH-1 in the tap withdrawal response (TWR) circuit premotor interneurons, AVA and AVD. Moreover, the phosphorylation of CRH-1 in these interneurons was required for normal LTM (Sugi, Ohtani, Kumiya, Igarashi, & Shirakawa, 2014).

Glutamatergic neurotransmission also plays an important role in LTM formation in C. elegans. Two genes involved in glutamate neurotransmission have been studied for their roles in LTM: eat-4 (glutamate vesicular transporter) and glr-1 (AMPA glutamate receptor subunit) (Rose, Kaun, & Rankin, 2002; Rose et al., 2003). In eat-4 mutant worms, spaced training with a tap stimulus did not result in LTM, but spaced training with a stronger stimulus, a train of taps, resulted in LTM (Rose, Kaun, & Rankin, 2002). It was hypothesized that the stronger stimuli are needed to cause the mechanosensory neurons’ release of sufficient glutamate to induce LTM in these eat-4 mutants.

Worms with mutations in glr-1 were also deficient in LTM formation (Rose et al., 2003). Confocal imaging of transgenic worms expressing GLR-1 tagged with GFP provided further evidence for GLR-1’s importance in LTM. When imaged 24 h after spaced training, the area of GLR-1::GFP expression of the worms decreased in the posterior ventral nerve cord region. These results support the hypothesis that LTM for tap habituation is possibly due to a decrease in strength of TWR circuit synapses. Additionally, a second type of AMPA glutamate receptor subunit, GLR-2, is involved in LTM for tap habituation (Emtage, Chang, Tiver, & Rongo, 2009). GLR-2 interacts with a synaptic scaffolding molecule, MAGI-1, in the premotor interneurons of the TWR circuit (Emtage, Chang, Tiver, & Rongo, 2009). glr-2 and magi-1 mutants were both LTM deficient when tested for mechanosensory memory 24 h after training. While GLR-1 and GLR-2 mainly contribute to the formation of a heteromeric AMPA-type receptor, GLR-1 homomers are less commonly found in C. elegans (Mellem, Brockie, Zheng, Madsen, & Maricq, 2002). The ubiquitination of GLR-1/GLR-2 heteromers results in their removal from the cell membrane and degradation—these cellular processes were observed to occur after LTM training (Burbea, Dreier, Dittman, Grunwald, & Kaplan, 2002; Emtage et al., 2009). Thus, MAGI-1, GLR-1, and GLR-2 mediate GLR-1/GLR-2 heteromers and contribute to LTM for tap habituation.

Intermediate-Term Memory (ITM) for Tap Habituation

Caenorhabditis elegans (C. elegans) can show intermediate-term memory (ITM) for tap habituation, which can be observed 12 h after a single massed training protocol of 80 taps at a 60-s interstimulus interval (ISI) (Li, Timbers, Rose, Bozorgmehr, McEwan, & Rankin, 2013) (Figure 3). Compared to long-term memory (LTM), ITM does not last for 24 h, is independent of protein synthesis, and worms with mutations in LTM genes are not ITM deficient. This means that LTM-deficient mutant strains such as crh-1, eat-4, and glr-1 showed normal ITM—suggesting that CREB activity and glutamate transmission are not involved in ITM. Intermediate-term memory was found to be dependent on a gene expressed in the mechanosensory neurons of the tap withdrawal circuit, flp-20, which encodes an FMRFamide-related neuropeptide. No ITM was seen in flp-20 worms, and ITM was rescued if flp-20 was expressed in only the mechanosensory neurons.

Context Conditioning for Tap Habituation

Caenorhabditis elegans (C. elegans) also show a form of chemosensory associative learning with tap habituation called context conditioning. This context conditioning was observed for both short-term memory (STM) and long-term memory (LTM). Short-term memory context-conditioning experiments were carried out by training worms with 30 taps at a 10-s interstimulus interval (ISI) in the presence/absence of a cue (e.g., the odorant, diacetyl). One hour later, the worms were tested in the presence/absence of the same cue. Worms given tap habituation training in the presence of a chemosensory cue (either a taste or smell) showed greater memory for habituation when tested in the presence of the same cue compared to worms that received different cues at training and testing (Rankin, 2000; Lau, Timbers, Mahmoud, & Rankin, 2013). Long-term memory context-conditioning experiments were conducted similarly, but training consisted of 5 blocks of 20 taps at a 10-s ISI in the presence/absence of an olfactory cue and testing consisted of 10 taps 24 h after training in the presence/absence of the same cue.

Three genes were shown to be involved in short and long-term memory for context conditioning: nmr-1 (a NMDA glutamate receptor subunit), glr-1 (an AMPA glutamate receptor subunit), and crh-1 (the transcription factor, CREB) (Lau et al., 2013). Short-term memory for context conditioning required nmr-1 and glr-1, while LTM for context conditioning required crh-1 in addition to nmr-1 and glr-1. The expression of NMR-1 in the pair of RIM interneurons in nmr-1 mutant worms was sufficient to rescue both STM and LTM for context-conditioning deficits. These results suggest that the RIM interneurons are a crucial site for the integration of chemosensory and mechanosensory input.

Tap Habituation to Aversive Stimuli

Previously, habituation was thought to be a process that allows animals to ignore unimportant repetitive stimuli. However, it is now recognized that habituation can also occur to aversive stimuli. Activation of a pair of polymodal nociceptor neurons in the nose of Caenorhabditis elegans (C. elegans), the ASH neurons, elicits a reversal response to physical contact, chemical repellents, and osmotic pressure (Bargmann, Thomas, & Horvitz, 1990; Hilliard, Bargmann, & Bazzicalupo, 2002; Hilliard, Apicella, Kerr, Suzuki, Bazzicalupo, & Schafer, 2005; Kaplan & Horvitz, 1993). Many of these stimuli are toxic and potentially lethal to the worm. The ASH neurons therefore play a role in the worm’s detection of threatening/toxic stimuli. A study examined habituation by repeatedly activating the ASH neurons, which expressed blue-light-activated ion channels (ChR2) (Ardiel, Yu, Giles, & Rankin, 2017). This stimulation resulted in the worms moving backward in the same way they do to natural ASH stimuli. Habituation of the ASH response also involved different patterns of plasticity for different components of the response. With repeated blue-light stimulation, response probability was maintained, response latency increased, and habituation was observed as a decrease in response duration. In addition, sensitization of forward locomotion between ASH stimulations was observed. These results suggest that habituation is not just a simple reduction of a response, but is part of an integration of plasticity in different response components (probability, duration and latency of the reversal response, and forward movement between stimuli) that optimize the animal’s ability to continue to respond to a toxic stimulus, while at the same time decreasing backward movement and increasing forward movement to disperse away from the site where the stimuli were received.

The plasticity of these ASH responses involves the pigment-dispensing factor receptor, PDFR (a member of the secretin receptor family which regulates mate-searching behavior) (Ardiel et al., 2017; Janssen et al., 2008). Worms without the pdfr-1 gene did not show the same increase in latency of the response as wild-type worms and they showed less forward movement in the intervals between stimuli than wild-type worms. The PDFR-1 receptor does not seem to affect the worm’s initial response to danger, but instead eliminates the changes seen in response to repeated ASH stimulation (Ardiel et al., 2017).

Chemosensory Learning and Memory

Introduction

Caenorhabditis elegans (C. elegans) can detect and move toward or away from a wide range of chemicals, which can be olfactory (e.g., diacetyl) or gustatory (e.g., sodium chloride): This behavior is called chemotaxis (Bargmann, 2006). It is most commonly measured by one of two assays, accumulation and tracking. Accumulation is measured by calculating the number of worms that amass at a chemical’s location, while tracking is evaluated by following the path of worms as they move toward or away from a chemical. Chemotaxis is associated with several forms of learning, including state-dependent learning, chemosensory classical conditioning, and aversive learning.

State-Dependent Learning

Caenorhabditis elegans (C. elegans) can show state-dependent learning, by making associations between its physiological state and environmental stimuli (Bettinger & McIntire, 2004). In the assay for state-dependent learning, worms pre-exposed to an olfactory cue in the presence of ethanol later associated this odor with their physiological state (i.e., sober, or inebriation induced by ethanol). When the worms were later exposed to the same cue, they only showed sensory adaptation if they were in the same physiological state as when they were pre-exposed. Worms with mutations that have impaired dopaminergic signaling are defective in state-dependent learning. This suggests that dopamine plays a role in state-dependent learning in C. elegans.

Chemosensory Classical Conditioning

Classical conditioning is a form of associative learning in which a neutral stimulus (conditioned stimulus, CS) is paired with another stimulus (unconditioned stimulus, US), which itself elicits an innate response. After the association between the CS and US is learned, the CS alone is enough to produce the automatic response that is normally elicited by the US. The paradigm used to study this form of learning in C. elegans involves first exposing worms to a US, which is either attractive (e.g., bacterial food source) or aversive (e.g., starvation), paired with a chemosensory CS. This results in the worms associating the CS with either attraction or aversion. Learning is observed in the trained worms when they have altered their preference for the CS compared to untrained worms. Food or starvation are most often used as attractive or aversive USs respectively. However, some studies have used hydrochloric acid as an aversive gustatory US (Amano & Maruyama, 2011) or potassium chloride as an appetitive gustatory US (Nishijima & Maruyama, 2017). Examples of olfactory CSs include diacetyl, butanone, 1-propanol, and 1-nonanol, while sodium chloride (NaCl) is commonly used as a gustatory CS. Similar to mechanosensory/tap habituation, Caenorhabditis elegans (C. elegans) shows both short- and long-term memory for classical conditioning. Short-term memory (STM) is induced by one block of training (single presentation of CS and US), while long-term memory (LTM) is induced by spaced training (multiple blocks of CS and US pairings) (Amano & Maruyama, 2011).

A number of genes have been found to be involved in associative learning and memory (STM, LTM) using classical conditioning paradigms. The genes lrn-1 and lrn2 were the first genes found to affect associative learning in C. elegans (Morrison, Wen, Runciman, & van der Kooy, 1999). In this study, wild-type worms learned to avoid a previously attractive odor, diacetyl (the CS), after it was paired with an aversive solution, acetic acid (the US). The lrn-1 and lrn-2 mutants were found to be defective in learning. The products of these two genes are currently unknown. In a later study using the same CS and US, glr-1 mutants were also found to be defective in associative learning (Morrison & van der Kooy, 2001). Using 1-propanol as the CS and hydrochloric acid as the US, wild-type and mutant worms were evaluated for STM and LTM of learned aversion to 1-propanol (Amano & Maruyama, 2011). The nmr-1 (a NMDA glutamate receptor subunit) mutants were both STM and LTM deficient, while glr-1 (an AMPA glutamate receptor subunit) and crh-1 (the transcription factor (CREB) mutants were LTM defective. In a similar study, but with 1-nonanol as the CS and potassium chloride as the US, nmr-1 mutants were again found to be both STM and LTM defective, while glr-1 and crh-1 mutants were LTM defective (Nishijima & Maruyama, 2017). A recent study, with butanone as the CS and food as the US, determined the AIM interneurons to be the location where CRH-1 activity is required for long-term associative memory (Lakhina et al., 2015). Through a genome-wide transcriptional analysis of CREB mutants, CRH-1 was also found to induce changes in gene expression for 757 target genes. In another study, with diacetyl as the CS and starvation as the US, add-1, which encodes the actin capping cytoskeletal protein α‎-adducin, was found to be required for STM and LTM of learned aversion (Vukojevic et al., 2012). During this memory formation, ADD-1 was also found to stabilize synapses at the AVA interneurons.

Another associative learning paradigm involves pairing an originally attractive taste or smell with starvation. As a result of the pairing, worms decreased their migration toward, or even avoided, the previously attractive stimulus. With this paradigm, it was found that CMK-1 (ortholog of mammalian calcium/calmodulin-dependent kinases, CAMKI/IV) acts in the ASE salt-sensing sensory neurons to mediate salt-aversive learning (Lim, Fehlauer, Glauser, Brunet, & Goodman, 2017). Another study elucidated some of the neural circuitry involved in aversive learning (Chen, Hendricks, Cornils, Maier, Alcedo, & Zhang, 2013) and showed that two insulin/insulin-like peptides (ILPs), ins-6 and ins-7, play antagonistic roles in olfactory aversive learning. The ILP ins-6 acts from the ASI sensory neurons to allow learning to occur, by repressing the transcription of ins-7 in the URX sensory neurons. Conversely, ins-7 disrupts learning by inhibiting RIA interneuron activity via DAF-2 (insulin receptor-like homolog) binding.

Aversive Learning

Caenorhabditis elegans Learning and MemoryClick to view larger

Figure 4. Aversive learning measured by an orientation test. Worms were suspended in a microdroplet and subjected to alternating air streams odorized with either OP-50 (non-pathogenic bacteria) or PA14 (pathogenic bacteria). A camera tracked the worm’s turning frequencies toward each other. Learning was seen when worms that had been exposed to PA14, later decreased their turning frequencies toward PA14, compared to worms that had not previously been exposed to PA14. The results are vertically arranged on raster plots where each dot represents one omega bend (a deep bend that is usually on the worm’s ventral side and changes the direction of its forward movement). Each row represents one cycle of the orientation test on one worm, and each worm was subjected to 12 cycles continuously in every test.

Adapted from Ha et al. (2010).

Caenorhabditis elegans (C. elegans) are also able to learn to avoid harmful stimuli. For instance, they will modify their preferences for odors after being exposed to pathogenic bacteria (Zhang, Lu, & Bargmann, 2005; Ha et al., 2010). Naïve/untrained worms are attracted to pathogenic bacteria (Ha et al., 2010). However, after exposure to the bacteria, worms learned to avoid the odors associated with them. Learning was measured by either a two-choice test or an orientation test. In the two-choice test, non-pathogenic and pathogenic bacteria were spotted on opposite sides of an agar plate. Worms were first placed at equal distances from the two spots of bacteria. One to two hours later, an accumulation of worms at one of the two spots was seen. Learning was determined by a decrease in the number of worms at the pathogenic bacteria spot. In the orientation test, worms were suspended in a microdroplet and subjected to alternating air streams odorized with pathogenic or non-pathogenic bacteria (Figure 4). A camera tracked the worms’ turning frequencies toward each odor. Learning was seen when worms that had been exposed to pathogenic bacteria, later decreased their turning frequencies toward pathogenic bacteria, compared to worms that had not previously been exposed to pathogenic bacteria.

Worms with mutations in tph-1 (enzyme, tryptophan hydroxylate, required for serotonin synthesis) and mod-1 (a serotonin-gated chloride channel) were found to be impaired in the pathogenic aversive-learning paradigm (Zhang et al., 2005). This suggests that serotonin (5-HT) plays a role in aversive learning.

Thermosensory Learning and Memory

Thermotaxis is the movement of an organism in response to temperature. If Caenorhabditis elegans (C. elegans) are cultivated in an environment at a specific temperature, they migrate toward that cultivation temperature when placed on a temperature gradient (Hedgecock & Russell, 1975). Conversely, if they experience starvation in an environment with a certain temperature, they move away from that temperature when placed in a new environment with a temperature gradient (Hedgecock & Russell, 1975). This was not originally recognized as learning; however after the publication of Rankin et al. (1990) researchers began to study it as learning.

The neural circuitry underlying thermotaxis consists of thermosensory neurons and interneurons (Mori & Ohshima, 1995) (Figure 5). The main thermosensory neurons, AFD and AWC, sense temperature information in the environment (Bargmann, Hartwieg, & Horvitz, 1993; Biron, Wasserman, Thomas, Samuel, & Sengupta, 2008; Kuhara et al., 2008). Both AFD and AWC neurons send signals to the interneuron AIY, which then sends information to the AIZ interneuron. The AIY interneuron mediates thermophilic movements, in which worms migrate toward warm temperatures. In contrast, AIZ mediates cryophilic movements, in which worms move toward colder temperatures. The RIA interneurons then integrate signals from AIY and AIZ, and send information to downstream interneurons and motor neurons to regulate movement.

Caenorhabditis elegans Learning and MemoryClick to view larger

Figure 5. A model of the neural circuitry underlying thermostatic learning and memory. The AFD and AWC neurons sense temperature information. Under certain conditions, the ASI chemosensory neurons are also involved in thermotaxis. The AIY interneuron receives information from the sensory neurons and sends information to the AIZ interneuron. Signals from both interneurons are then transmitted to the RIA interneurons. Solid lines represent synaptic connections while dashed arrows represent secretory communications.

Adapted from Aoki and Mori (2015).

Through forward genetic screens a number of genes important for thermotaxis and for thermotaxic learning and memory have been identified; three of the genes that affect the ability to learn the association between temperature and the presence or absence of food are aho-1 (abnormal hunger orientation), aho-2/ins-1, and aho-3 (Mohri, Kodama, Kimura, Koike, Mizuno, & Mori, 2005; Kodama et al., 2006; Nishio et al., 2012). The first of these, aho-1, has not been genetically mapped, so little is known about its function. The second, aho-2/ins-1, encodes an insulin-like peptide orthologous to human insulin; the encoded peptide is one of the 38 insulin-like peptides in C. elegans (Kodama et al., 2006). The third, aho-3, encodes a protein homologous to the protein FAM108B1, which is expressed in the human brain; FAM108B1 shares highly conserved molecular properties with AHO-3 (Nishio et al., 2012). When these mutant worms were well fed, they showed normal thermotaxis; they properly associated a temperature with the presence of food. However, these worms did not learn to avoid the cultivation temperature if they were raised in a starved state. These behaviors suggested that the mutants had deficits in learning the association between temperature and their fed or starved state, even though they showed normal temperature sensing and encoding.

Long-term memory (LTM) for thermotaxis has also been shown in C. elegans (Nishida, Sugi, Nonomura, & Mori, 2011). Similar to chemosensory classical conditioning and LTM for mechanosensory habituation, LTM for thermotaxis requires crh-1 (homolog of the transcription factor, CREB) (Nishida et al., 2011). When crh-1 was rescued in the thermosensory neurons AFD, the worms showed normal LTM. Thermosensory LTM also requires RCAN-1, a calcipressin that inhibits calcineurin, TAX-6 in the AFD interneurons (Li, Bell, Ahnn, & Lee, 2015).

Learning and Memory in Other Sensory Modalities: Oxygen and Carbon Dioxide

Oxygen

Caenorhabditis elegans Learning and MemoryClick to view larger

Figure 6. Caenorhabditis elegans (C. elegans) stores a memory of previous O2 experience primarily in the neural circuit shown. Arrows denote chemical synapses, while bars denote gap junctions. The strength and type (excitatory or inhibitory) of the synapses are not indicated. Sensory neurons are indicated with blue rectangles, interneurons with red, motor neurons with a green diamond, and motor output with a light-blue hexagon.

Adapted from Metaxakis, Petratou, and Tavernarakis (2018).

Oxygen preference in Caenorhabditis elegans (C. elegans) has been shown to be altered by experience (Cheung, Cohen, Rogers, Albayram, & de Bono, 2005). When worms were cultivated in hypoxic conditions (1% O2) for 4–6 h, they migrated to the hypoxic area of 0-7% when placed in an oxygen gradient of 0–21%. This behavior indicates that the worms have memory for the oxygen environment they were cultivated in—this is primarily mediated by the sensory neurons, AQR, PQR, and URX (Cheung et al., 2005) (Figure 6). When oxygen levels drop, the soluble guanylate cyclases, GCY-35 and GCY-36, appear to become activated and depolarize these three sensory neurons. It has been suggested that C. elegans stores this memory of oxygen experience in a circuit involving RMG interneurons, which are connected to URX sensory neurons by gap junctions and synapses (Fenk & de Bono, 2017).

Carbon Dioxide

Carbon dioxide avoidance in C. elegans has been shown to be altered by experience with food and oxygen (Bretscher, Busch, & de Bono, 2008; Hallem & Sternberg, 2008). Starvation in worms strongly suppressed carbon dioxide avoidance—this is regulated by the BAG sensory neurons, the cGMP-gated heterodimeric channel TAX-2/TAX-4, and many other signaling molecules including the neuropeptide Y receptor, NPR-1 (Hallem & Sternberg, 2008). Exposure to hypoxic conditions (<1% O2) also suppressed carbon dioxide avoidance (Bretscher et al., 2008). The transcription factor, HIF-1, mediates the oxygen and carbon dioxide pathways, suggesting that these pathways interact in an antagonistic manner (Bretscher et al., 2008).

Conclusion

Caenorhabditis elegans (C. elegans) exhibits remarkable behavioral plasticity, as it is capable of displaying many forms of learning and memory within multiple sensory modalities, including mechanosensation, chemosensation, and thermosensation. Several important genes and neural circuits responsible for these behaviors have been identified and have thereby shed light on some of the mechanisms underlying learning and memory in C. elegans.

Its genetic amenability, mapped nervous system, and the myriad of available biological tools available to it are just a few of the many characteristics of C. elegans that make it a powerful model organism for the study of learning and memory. As there is increasing evidence showing that the mechanisms of plasticity in C. elegans can be generalized to more complex organisms, the use of C. elegans for research will continue to contribute to our understanding of the foundational mechanisms of learning and memory.

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