Author response: AMPK acts as a molecular trigger to coordinate glutamatergic signals and adaptive behaviours during acute starvation

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract The stress associated with starvation is accompanied by compensatory behaviours that enhance foraging efficiency and increase the probability of encountering food. However, the molecular details of how hunger triggers changes in the activity of neural circuits to elicit these adaptive behavioural outcomes remains to be resolved. We show here that AMP-activated protein kinase (AMPK) regulates neuronal activity to elicit appropriate behavioural outcomes in response to acute starvation, and this effect is mediated by the coordinated modulation of glutamatergic inputs. AMPK targets both the AMPA-type glutamate receptor GLR-1 and the metabotropic glutamate receptor MGL-1 in one of the primary circuits that governs behavioural response to food availability in C. elegans. Overall, our study suggests that AMPK acts as a molecular trigger in the specific starvation-sensitive neurons to modulate glutamatergic inputs and to elicit adaptive behavioural outputs in response to acute starvation. https://doi.org/10.7554/eLife.16349.001 eLife digest Animals often need to adapt to changes in food availability in order to survive. When food is in short supply and animals are starving, their energy reserves are low. To conserve energy, behaviours that are not essential to survival, like mating, are put on hold. Instead, animals channel their energies into foraging strategies that may help them find new food sources. These behavioural changes are likely to be caused by changes in brain activity triggered by starvation. It is not entirely clear how starvation changes the brain and consequently how an animal behaves. It is also difficult to study how the brain regulates behaviour in response to environmental changes like food availability in larger animals with more complex nervous systems. Instead, scientists often study less complex animals like a type of worm called C. elegans, because this model organism has a simpler nervous system and it is easier to observe its feeding behaviours. Previous observations have revealed that well-fed worms travel backwards when they are hungry, revisiting sites where they have previously found food. Yet, when the worms are starving, they move forward more frequently, presumably to find new sources of food. Now, Ahmadi and Roy show starving worms activate an enzyme called AMP-activated protein kinase (or AMPK for short). Worms genetically engineered to lack this enzyme tend to move backward when they are starved, instead of moving forward like typical starving worms. This shows that AMPK triggers a wider search for new food sources. Further experiments showed that AMPK acts to inhibit two receptors, which in turn, affects the activity of two different neurons. These two neurons work together to change the animal's behaviour and boost the likelihood the animal will be able to find a new food source when food is scarce. More complex animals, including humans, also have the receptors and brain cells targeted by AMPK in response to starvation. Future studies are needed to determine whether a similar chain of events occurs in creatures with more complicated nervous systems. https://doi.org/10.7554/eLife.16349.002 Introduction Most organisms are faced with unpredictable fluctuations in their natural environment that often lead to periods of limited food resources. Their ability to adapt to these changes in resource availability is critical for survival and is often a driving force in evolution (Gray et al., 2004; Wang et al., 2005). When resources are scarce, pathways associated with energy conservation at both the organismal and the cellular levels become activated, and these are often complemented by behavioural modifications that simultaneously enhance foraging efficiency (Wang et al., 2005; Ashrafi, 2006). The mammalian central nervous system (CNS) integrates internal and external cues that signal energy demand and availability and coordinately regulates outputs ranging from energy expenditure to feeding and associated locomotory behaviours (Cone, 2005; Balthasar et al., 2005; Belgardt et al., 2009; Dietrich and Horvath, 2011; Yang et al., 2011; Aponte et al., 2011; Sternson et al., 2013; Dietrich et al., 2015). In mice, the Agouti-related protein (AGRP)- and Pro-opiomelanocortin (POMC)-expressing neurons in the arcuate nucleus of the hypothalamus form a core circuit to regulate food intake and energy expenditure through the modulation of their neuronal activity in response to hormonal signals linked to metabolic status (Cowley et al., 2001; Bewick et al., 2005; Yang et al., 2011). The activity of both of these neuronal populations is mediated by engaging signalling pathways that control the strength and/or plasticity of rapid, excitatory glutamatergic transmission (Bito et al., 2010; Collingridge et al., 2010; Liu et al., 2012), but how energy stress results in changes in neuronal activity to elicit adaptive, or even compulsive behaviours are just now beginning to be elucidated (Dietrich et al., 2015). The many signalling networks that are triggered throughout the nervous system that mediate the action of small molecules, hormones and nutrients on energy balance are of major interest due to their implication in, or treatment of various disorders. One of the key factors of paramount importance for metabolic homeostasis and survival is a highly conserved heterotrimeric protein kinase called AMP-activated protein kinase (AMPK). AMPK is regulated by the ratio of cellular AMP/ATP and by upstream activating kinases (Hardie, 2008; Hardie et al., 2012). It functions as a 'fuel gauge' to monitor cellular energy status by inhibiting anabolic pathways and activating catabolic pathways so as to generate sufficient levels of metabolic substrates required to maintain a minimal threshold of basal cellular activities (Hardie, 2008; Hardie et al., 2012; Hardie and Ashford, 2014) Energy stress has been demonstrated to induce adaptive behaviours in a neuronal AMPK-dependent manner (Lee et al., 2008; Cunningham et al., 2014). Moreover, accumulating evidence has implicated AMPK in the hypothalamic regulation of metabolic rate and food intake behaviour (Kola, 2008; Lopez et al., 2010; Lim et al., 2010; Yang et al., 2011; Schneeberger and Claret, 2012). However, our understanding of how starvation influences adaptive foraging behaviours in an AMPK-dependent manner is still largely unknown, mostly due to the overwhelming complexity of the response in higher animals (Dietrich et al., 2015). In C. elegans these foraging behaviours are comparatively simple, consisting of a series of forward or backward movements, specific turns, or changes in direction (Gray et al., 2005; Piggott et al., 2011; Chen et al., 2013; Hendricks, 2015). Food availability has been demonstrated to affect various aspects of these key elements in C. elegans locomotion (Sawin et al., 2000; Gray et al., 2005; Chalasani et al., 2007; Flavell et al., 2013). In the absence of food, well-fed animals reverse frequently, a behavioural pattern that reflects a sensory memory of food that is expressed by the navigation circuit and results in efficient exploration of a limited area. In contrast, starvation suppresses reversals and induces forward movement (runs) that allow animals to explore distal areas; a strategy that is referred to as dispersal behaviour or alternatively, distal exploration (Gray et al., 2005). The simplicity of the neural circuits and locomotory behaviours in conjunction with its amenability to genetic manipulation makes C. elegans an ideal model to investigate the mechanisms through which AMPK regulates neuronal activity and adaptive locomotory behaviour (searching for food) in response to hunger. Unlike most other organisms studied to date, C. elegans mutants that lack all AMPK signalling are viable, but show clear phenotypes when subjected to energy stress (Narbonne and Roy, 2006; 2009). Therefore, using calcium imaging, cell type–specific optogenetic techniques, and classic genetic analysis, we identified and characterized the neural circuit in which AMPK functions as a molecular switch. This circuit includes the AIB and AIY interneurons; two neurons that form one of the primary circuits that dictate appropriate food- and odour-evoked behaviours (Gray et al., 2005; Chalasani et al., 2007). We discovered that AMPK modulates AIB and AIY activity through two distinct mechanisms to ultimately ensure that adaptive foraging behaviours are appropriately triggered during periods of starvation. In the AIB interneurons AMPK regulates the abundance of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor GLR-1 in the postsynaptic elements, presumably by phosphorylation of serine 907 and 924 resulting in changes in synaptic strength and specific behavioural outputs. In addition, we also demonstrate that AMPK modulates a key metabotropic glutamate receptor called MGL-1 in the AIY interneuron at both mRNA and protein levels leading to an increase in AIY neuronal activity in starved animals. Together, our results indicate that AMPK acts as a starvation-inducible molecular trigger in the nervous system that modulates glutamatergic neuronal activity by at least two distinct mechanisms to modify behavioural outcomes in response to energy stress. Results Neuronal AMPK signalling triggers distal exploratory behaviour in starved animals AMPK has been implicated in the regulation of feeding behaviours in higher animals (Minokoshi et al., 2004; Yang et al., 2011) although its essential role in development and cellular homeostasis has made it very difficult to study its role outside this closed circuit. Accordingly, the identification of the neuronal targets of this protein kinase that may be important for these behaviours has been virtually impossible to interrogate. Fortunately, C. elegans mutants that completely lack AMPK signalling are viable, thus providing us with an opportunity to investigate how AMPK regulates characteristic foraging behaviours in response to acute periods of starvation. We and others found that removal of aak-2, the more prominent of two catalytic subunits of AMPK present in C. elegans, disrupts most cellular AMPK signalling and affects various aspects of C. elegans foraging behaviour (Lee et al., 2008; Cunningham et al., 2014). More specifically, aak-2 mutants exhibit a behavioural profile in response to acute starvation that is more typical of satiated animals, where animals tend to explore locally rather than foraging in more distant locations. This is reflected by the frequency of body bends; which is considered as a representative readout for locomotory movement away from a nutrient-depleted environment in search of new resources, and reversals; a behaviour most often associated with local exploration (Gray et al., 2005). Both of these behaviours are affected in starved aak-2 mutants (Figure 1A). These behaviours appear to be regulated predominantly by aak-2 activity since removal of both catalytic subunits and hence eliminating all AMPK signalling (aak(0)) only modestly enhanced the phenotype of aak-2 mutants in all our behavioural assays suggesting that aak-2 mutants recapitulate a severe loss of AMPK function. Figure 1 with 2 supplements see all Download asset Open asset Neuronal AMPK regulates distal exploratory behaviour in starved animals. (A) Starved aak-2 mutants display defective transition from local to distal exploration indicated by (i) decreased forward locomotion and (ii) increased reversal rate during acute bouts of starvation. These phenotypes are not dependent on aak-1 (n>40), (one-way ANOVA **p<0.001, ***p<0.0001). (B) AMPK mutants display persistent decreased forward locomotion rate (i) and increased reversal frequency (ii) during periods of acute starvation (n>10), Error bars represent ± SEM (Student's t-test, *p<0.05, **p<0.001, ***p<0.0001). (C) aak-2 reconstitution within the nervous system using the pan-neural [Punc-119::aak-2] transgene rescues the defective distal exploratory behaviour in starved aak-2 mutants while its reconstitution within the body wall muscle using [Punc-54::aak-2] transgene does not improve the distal exploratory behaviour of aak-2 mutants (n>15), (one-way ANOVA *p<0.05, **p<0.001). (D,E,F) aak-2 is highly expressed throughout the nervous system. Scale bars are 40 μm. In the box and whisker plots (A,C) the central line is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points. https://doi.org/10.7554/eLife.16349.003 Figure 1—source data 1 Locomotory behaviour in AMPK mutant animals. https://doi.org/10.7554/eLife.16349.004 Download elife-16349-fig1-data1-v1.xlsx Figure 1—source data 2 Food and stimuli-related behaviours in AMPK mutants. https://doi.org/10.7554/eLife.16349.005 Download elife-16349-fig1-data2-v1.xlsx Figure 1—source data 3 Locomotory behaviour upon depletion of aak-2 with 1535 RNAi. https://doi.org/10.7554/eLife.16349.006 Download elife-16349-fig1-data3-v1.xlsx Since aak-2 is a major regulator of foraging behaviour in C. elegans, we transferred mid-fourth larval stage (L4) aak-2 mutant larvae to bacteria-free plates and monitored their movements for 20 hr to delineate sub-behaviours underlying aak-2 deficiency. We found that starved aak-2 mutants show defects in their rate of forward locomotion measured in the absence of food, which is compounded by their inability to appropriately suppress reversal behaviour during periods of more prolonged starvation (Figure 1B). Because we observe changes in each of these AMPK-dependent parameters in starved animals, and given the antagonistic relationship between forward and backward locomotion where increased reversal frequency affects the onset and/or duration of forward movement (Burbea et al., 2002; Juo et al., 2007), we chose to assess both behaviours in starved animals throughout our study. The aak-2-dependent foraging defects we observed in starved animals are not likely to be due to a more general role of AMPK in the regulation of appropriate motor neuron development or function since AMPK mutants were comparable to wild type controls when reversal frequency and forward locomotion were quantified during conditions when food was abundant (Figure 1—figure supplement 1A). Moreover, aak-2 mutants displayed a normal fleeing speed in response to a mechanical stimulus that was applied to the posterior (Figure 1—figure supplement 1D). In addition, we observed that aak-2 (RNAi) performed at the late L2 stage, when the majority of neurons have already been generated, resulted in a reduction in forward locomotion compounded with an increase in reversal frequency in starved animals. This suggests that the reduced locomotory speed is unlikely to be the consequence of a general defect in motor function or neuromuscular development (Figure 1—figure supplement 2). To discern whether aak-2 is specifically required in neurons, muscle, or both tissues to modulate distal exploratory behaviours we used tissue-specific promoters to drive aak-2 expression and test its ability to correct the behavioural defects typical of AMPK mutants. While an aak-2 cDNA (Narbonne and Roy, 2009) expressed under the control of a pan neuronal promoter [Punc-119::aak-2] rescued the distal exploratory defect of aak-2 mutants, aak-2 mutants continued to exhibit prolonged local exploration when the same aak-2 cDNA was reconstituted in body wall muscle using [Punc-54::aak-2] transgene (Figure 1C). Consistent with these results, we observed that a rescuing translational fusion reporter [Paak-2::aak-2::GFP] was broadly expressed throughout the nervous system (Figure 1D,E,F). These data are consistent with previous observations indicating that AMPK is required in the nervous system to regulate locomotory behaviour in response to food availability (Lee et al., 2008; Cunningham et al., 2014). AMPK is not required for all starvation-related behaviours The ability of AMPK mutants to appropriately respond to starvation could result from a global AMPK-dependent defect in their nervous system rendering them incapable of responding to multiple cues in addition to starvation. However, this seems unlikely since two other food-related behaviours namely, basal slowing response and enhanced slowing response, which are triggered in well-fed and starved animals that are reintroduced to food, respectively, remain unaffected in AMPK mutants. In its natural habitat, a well-fed animal would be more likely to risk exploring distant locations for high quality food sources, whereas a starved animal would be less likely to stray far from a recently discovered vital food supply. These paradigms regulate behavioural plasticity in response to starvation in C. elegans and are mediated by distinct dopaminegic and serotonergic signalling (Sawin et al., 2000). When we re-introduced well-fed or starved aak-2 mutants to food we did not detect any significant difference in either the basal slowing response or the enhanced slowing response (Figure 1—figure supplement 1B,C) suggesting that AMPK is not involved in the behavioural plasticity that occurs after re-introduction of animals to food and that the neural circuitry that mediates such behavioural plasticity is presumably intact and functional in animals that lack AMPK. AAK-2 is required in both the AIY and the AIB interneurons to mediate the transition from local to distal exploration in response to starvation Since pan neuronal aak-2 expression rescued the distal exploration defect of aak-2 mutants, we next sought to identify the individual neurons that require aak-2 activity to trigger this behaviour. The neural circuitry that dictates these simple behavioural outcomes has been well described (White et al., 1976, 1986; Chalfie et al., 1985; Wicks et al., 1996; Gray et al., 2005). In particular, five pairs of command interneurons are required for the control of coordinated movement. The PVC and AVB interneurons are primarily required for the initiation of forward movement, while the AVA, AVD, and AVE interneurons control reversals (White et al., 1976; Chalfie et al., 1985; Wicks et al., 1996; Gray et al., 2005). Sensory inputs and serotonergic signalling together contribute to the behavioural changes that occur following removal from food (Gray et al., 2005). Moreover, several studies have underscored a critical role for both the AIY and the AIB interneurons, which are independently required to suppress and enhance reversals, respectively, resulting in longer or shorter durations of forward movement (Tsalik and Hobert, 2003; Wakabayashi et al., 2004; Gray et al., 2005; Chalasani et al., 2007; Luo et al., 2014). The RIM interneurons have also been determined as regulators of reversal frequency (Gray et al., 2005; Gordus et al., 2015). To map the precise sites of AMPK function in its regulation of distal exploratory behaviour, we introduced aak-2 in different subsets of neurons using neuronal sub-type-specific promoters: tph-1 (serotonergic neurons) (Cunningham et al., 2012), glr-1 (command interneurons) (Zheng et al., 1999), che-2 (chemosensory neurons) (Gray et al., 2005), ttx-3 (AIY) (Chalasani et al., 2007), npr-9(AIB) (Piggott et al., 2011), tdc-1 (RIM) (Cunningham et al., 2012) and rig-3 (AVA) (Marvin et al., 2013). Reconstitution of AMPK (aak-2) using the glr-1, tdc-1, ttx-3, npr-9 promoters was sufficient to partially rescue the defective distal exploration typical of aak-2 mutants suggesting that AMPK functions within these neurons to phosphorylate targets involved in regulating these behavioural responses. Conversely, introducing AMPK (aak-2) in the sensory neurons, the serotonergic neurons, or the command interneurons involved in triggering reversals failed to correct the defect (Figure 2A). As AMPK expression within the GLR-1-expressing neurons partially rescued the locomotory defect of aak-2 mutants, it is very likely that AMPK may be independently required in the GLR-1-expressing neurons AIB and RIM, although this does not exclude a role for aak-2 in other GLR-1-expressing neurons. Figure 2 with 1 supplement see all Download asset Open asset AMPK acts in the AIB and AIY interneurons to integrate chemosensory signals and trigger distal exploratory behaviour in starved animals. (A) Targeted expression of aak-2 within the GLR-1-expressing neurons [Pglr-1::aak-2], RIM neurons [Ptdc-1::aak-2], AIB interneurons [Pnpr-9::aak-2] and AIY interneurons [Pttx-3::aak-2], but not chemosensory neurons [Pche-2::aak-2], serotonergic neurons [Ptph-1::aak-2] or AVA neurons [Prig-3::aak-2] partially restores the distal exploratory defect in starved aak-2 mutants by affecting both forward locomotion (i) and reversal frequency (ii) (n>20), (one-way ANOVA *p<0.05, **p<0.001, ***p<0.0001). (B) Simultaneous rescue of aak-2 in the AIB and AIY using [Pnpr-9::aak-2][Pttx-3::aak-2] transgenes is sufficient to completely restore the defective exploratory behaviour typical of starved aak-2 mutants (n>20), (one-way ANOVA ***p<0.0001). (C) Starved worms were placed 1.5 cm away from a spot of food (fresh OP50) and the required to the food was monitored for the worms that found food within aak-2 mutants display defective food indicated by increased to find food and this defect be rescued by specific expression of aak-2 throughout the nervous system using [Punc-119::aak-2] transgene Targeted expression of aak-2 within the AIB and AIY interneurons, but not the chemosensory neurons rescue the defective of aak-2 mutants Error bars in represent ± SEM (one-way ANOVA *p<0.05, **p<0.001). Figure data 1 AMPK reconstitution in different neurons to restore the defective distal exploratory behaviour and defective typical of aak-2 mutants. Download The AIY and AIB interneurons from the sensory neurons and in to suppress and enhance reversals, respectively, in response to food availability (Figure supplement (Chalasani et al., 2007). The AIB interneurons their signals to the RIM and AVA, which in their outputs to the (Figure supplement et al., 2015). As an AMPK expression within the AIB and RIM partially the defective distal exploration of starved aak-2 mutants, it is likely that AMPK functions in multiple neurons in a circuit to regulate locomotory behaviour in response to starvation. When we expressed aak-2 in both the AIY and the AIB interneurons, we completely the defective distal exploration of aak-2 mutants, while expression of aak-2 within the RIM and the AIB interneurons, or in RIM and did not improve the distal exploration defect of starved aak-2 mutants the rescue observed in AMPK mutant animals aak-2 within the AIB or the AIY interneurons (Figure The RIM neurons form and with AIB and (White et al., it has been that from the RIM neurons enhance the in response to in the circuit et al., 2015). Therefore, aak-2 expression within the RIM neuron this the expression of aak-2 within the AIB and RIM neurons the circuit either a more or our data a critical role of AMPK in the AIY and AIB interneurons to regulate the appropriate transition from local to distal exploration in animals subjected to acute starvation. AMPK functions within the AIB and AIY interneurons to integrate sensory signals in response to starvation AMPK mutants demonstrate reduced forward movement accompanied by an increased frequency of these defects distal exploratory foraging behaviour starved aak-2 mutants could also be defective in food at a We placed starved animals on plates with a food source and monitored the required for each animal to the We that aak-2 mutants were less efficient than wild type controls in their ability to to the food source (Figure However, from this we were to this defect is a consequence of a in their ability to food or to integrate the of signals to appropriately modify locomotory behaviour since we also observed a significant defect in the ability of AMPK mutants to the (Figure However, since the expression of aak-2 within the sensory neurons failed to restore the defective distal exploratory behaviour in aak-2 mutants, while this defect was rescued by driving aak-2 expression specifically within the AIB and AIY interneurons, we that AMPK is critical for the of sensory cues and not with (Figure our results indicate that AMPK is required in the AIB and AIY interneurons to trigger appropriate exploratory behaviour in response to acute starvation. AMPK functions upstream of glr-1 and to modulate distal exploration To how and where AMPK functions within the AIY and AIB interneurons to modulate the transition between local and distal we performed optogenetic experiments to AIB and AIY activity in starved aak-2 mutants et al., 2012). of the AIY interneurons in aak-2 mutant animals resulted in a significant in reversal frequency that was persistent throughout the In contrast, AIY in animals the forward locomotion and reversal rate which is consistent with previous studies the of the AIY interneurons in starved animals (Gray et al., 2005; Flavell et al., (Figure Figure supplement 2A). Figure 3 with 1 supplement see all Download asset Open asset AMPK regulates glutamatergic of AIY interneurons or of AIB interneurons (B) in starved aak-2 mutants in the of induces distal exploration by reversals and forward locomotion (n>15), Error bars represent ± SEM ANOVA *p<0.05, ***p<0.0001). glr-1 or function rescues the defective exploratory behaviour of starved aak-2 mutants by forward locomotion (i) and reversals (ii) (n>20), (one-way ANOVA *p<0.05, **p<0.001, ***p<0.0001). In the box and whisker plots the central line is the median, the edges of the box are the 25th and 75th percentiles, and the whiskers extend to the most extreme data points. Figure data 1 and Download Figure data 2 The of locomotory behaviour of AMPK Download Figure data 3 AMPK phosphorylation targets expressed within the AIB and AIY Download In a we the behaviour of starved aak-2 mutants following of AIB by the et al., 2012). The of AIB was sufficient to suppress reversals in the aak-2 mutants, while also normal distal exploratory behaviour during starvation. In contrast, AIB in animals their locomotory behaviour which be by of AIB during acute bouts of starvation as the locomotory behaviour of starved animals has been to remain upon AIB (Gray et al., (Figure We these observations to indicate that although AMPK activity is required within the AIB and AIY interneurons for appropriate distal exploration in response to acute starvation, it does so normal neuronal development or within the identified the interneurons in which aak-2 is required to suppress reversals, forward and trigger distal we to identify AMPK targets within these interneurons that mediate its in response to starvation. we the C. elegans for that AMPK phosphorylation sites and were to be expressed in the AIB or AIY interneurons,

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