Different stressors uniquely affect the expression of endocannabinoid-metabolizing enzymes in the central ring ganglia of Lymnaea stagnalis.

The endocannabinoid system (ECS) plays an important role in neuroprotection, neuroplasticity, energy balance, modulation of stress, and inflammatory responses, acting as a critical link between the brain and the body's peripheral regions, while also offering promising potential for novel therapeutic strategies. Unfortunately, in humans, pharmacological inhibitors of different ECS enzymes have led to mixed results in both preclinical and clinical studies. As the ECS has been highly conserved throughout the eukaryotic lineage, the use of invertebrate model organisms like the pond snail Lymnaea stagnalis may provide a flexible tool to unravel unexplored functions of the ECS at the cellular, synaptic, and behavioral levels. In this study, starting from the available genome and transcriptome of L. stagnalis, we first identified putative transcripts of all ECS enzymes containing an open reading frame. Each predicted protein possessed a high degree of sequence conservation to known orthologues of other invertebrate and vertebrate organisms. Sequences were confirmed by qualitative PCR and sequencing. Then, we investigated the transcriptional effects induced by different stress conditions (i.e., bacterial LPS injection, predator scent, food deprivation, and acute heat shock) on the expression levels of the enzymes of the ECS in Lymnaea's central ring ganglia. Our results suggest that in Lymnaea as in rodents, the ECS is involved in mediating inflammatory and anxiety-like responses, promoting energy balance, and responding to acute stressors. To our knowledge, this study offers the most comprehensive analysis so far of the ECS in an invertebrate model organism.

Investigating the interactions between multiple memory stores in the pond snail Lymnaea stagnalis.

The pond snail Lymnaea stagnalis exhibits various forms of associative learning including (1) operant conditioning of aerial respiration where snails are trained not to open their pneumostome in a hypoxic pond water environment using a weak tactile stimulus to their pneumostome as they attempt to open it; and (2) a 24 h-lasting taste-specific learned avoidance known as the Garcia effect utilizing a lipopolysaccharide (LPS) injection just after snails eat a novel food substance (carrot). Typically, lab-inbred snails require two 0.5 h training sessions to form long-term memory (LTM) for operant conditioning of aerial respiration. However, some stressors (e.g., heat shock or predator scent) act as memory enhancers and thus a single 0.5 h training session is sufficient to enhance LTM formation lasting at least 24 h. Here, we found that snails forming a food-aversion LTM following Garcia-effect training exhibited enhanced LTM following operant condition of aerial respiration if trained in the presence of the food substance (carrot) they became averse to. Control experiments led us to conclude that carrot becomes a 'sickness' risk signal and acts as a stressor, sufficient to enhance LTM formation for another conditioning procedure.

A translational and multidisciplinary approach to studying the Garcia effect, a higher form of learning with deep evolutionary roots.

Animals, including humans, learn and remember to avoid a novel food when its ingestion is followed, hours later, by sickness - a phenomenon initially identified during World War II as a potential means of pest control. In the 1960s, John Garcia (for whom the effect is now named) demonstrated that this form of conditioned taste aversion had broader implications, showing that it is a rapid but long-lasting taste-specific food aversion with a fundamental role in the evolution of behaviour. From the mid-1970s onward, the principles of the Garcia effect were translated to humans, showing its role in different clinical conditions (e.g. side-effects linked to chemotherapy). However, in the last two decades, the number of studies on the Garcia effect has undergone a considerable decline. Since its discovery in rodents, this form of learning was thought to be exclusive to mammals; however, we recently provided the first demonstration that a Garcia effect can be formed in an invertebrate model organism, the pond snail Lymnaea stagnalis. Thus, in this Commentary, after reviewing the experiments that led to the first characterization of the Garcia effect in rodents, we describe the recent evidence for the Garcia effect in L. stagnalis, which may pave the way for future studies in other invertebrates and mammals. This article aims to inspire future translational and ecological studies that characterize the conserved mechanisms underlying this form of learning with deep evolutionary roots, which can be used to address a range of different biological questions.

Prey populations with different predation histories show differences in behavioral and transcriptional effects under acute predation threat.

Predator detection induces both behavioral and physiological responses in prey organisms. Our model organism, the pond snail Lymnaea stagnalis, shows multiple defensive behaviors in response to predator cues. In this study, we investigated and compared the transcriptional effects induced by the exposure to a predator scent (i.e., crayfish effluent - CE) in a strain of lab-inbred snails (i.e., W snails), which have been raised and maintained under standardized laboratory conditions for generations and a strain of freshly collected snails (i.e., Margo snails), which live in a crayfish-free pond. Neither the W- strain nor the Margo Lake snails used in this study have actually experienced crayfish. However, the W strain innately recognizes crayfish as a threat. We found that, following the exposure to CE, both strains showed significantly higher mRNA levels of serotonin-related genes. This is important, as the serotonergic system modulates predator detection and vigilance behaviors in pond snails. However, the expression levels of CREB1 and HSP70 were only upregulated in CE-exposed W snails but not in Margo ones. As CREB1 plays a key role in learning and memory formation, whereas HSP70 is involved in stress response, we investigated whether these differences in CREB1 and HSP70 mRNA levels would reflect differences in predator-induced learning (e.g., configural learning). We found that only W snails formed configural learning memory, whereas Margo snails did not. Thus, while both the strains molecularly respond to the CE by upregulating the serotoninergic system, only W snails behaviorally recognize CE as a threat and, therefore, form configural learning.

Gut microbiota alterations promote traumatic stress susceptibility associated with p-cresol-induced dopaminergic dysfunctions.

Mounting evidence suggests a link between gut microbiota abnormalities and post-traumatic stress disorder (PTSD). However, whether and how the gut microbiota influences PTSD susceptibility is poorly understood. Here using the arousal-based individual screening model, we provide evidence for pre-trauma and post-trauma gut microbiota alterations in susceptible mice exhibiting persistent PTSD-related phenotypes. A more in-depth analysis revealed an increased abundance of bacteria affecting brain processes including myelination, and brain systems like the dopaminergic neurotransmission. Because dopaminergic dysfunctions play a key role in the pathophysiological mechanisms subserving PTSD, we assessed whether these alterations in gut microbiota composition could be associated with abnormal levels of metabolites inducing dopaminergic dysfunctions. We found high levels of the l-tyrosine-derived metabolite p-cresol exclusively in the prefrontal cortex of susceptible mice. We further uncovered abnormal levels of dopamine and DOPAC, together with a detrimental increase of dopamine D3 receptor expression, exclusively in the prefrontal cortex of susceptible mice. Conversely, we observed either resilience mechanisms aimed at counteracting these p-cresol-induced dopaminergic dysfunctions or myelination-related resilience mechanisms only in the prefrontal cortex of resilient mice. These findings reveal that gut microbiota abnormalities foster trauma susceptibility and thus it may represent a promising target for therapeutic interventions.

Behavioral and Transcriptional Effects of Short or Prolonged Fasting on the Memory Performances of Lymnaea stagnalis.

INTRODUCTION: The Garcia effect, a solid learning paradigm, was used to investigate the molecular and behavioral effects induced by different lengths of fasting on the cognitive functions in the pond snail Lymnaea stagnalis, a valid model system. METHODS: Three experimental groups were used: moderately hungry snails, food-deprived for 1 day (D1 snails), severely hungry snails (D5 snails), fasting for 5 days, and satiated snails with ad libitum access to food (AL snails). In the Garcia effect, a single pairing of an appetitive stimulus with a heat stressor results in a learned taste-specific negative hedonic shift. D5 snails were injected with bovine insulin and D1 snails with the insulin receptor antibody (Ab). As a control group, AL snails were injected with saline. Gene expression analyses were performed by real-time PCR in snails' central nervous system (CNS). RESULTS: AL snails are "average learners," D1 snails are the best performers, whereas the D5 ones do not show the Garcia effect. Severely fasting snails injected with insulin 3 h before the training procedure show the Garcia effect, whereas injecting 1-day fasting snails with insulin receptor Ab blocks their ability to express memory. The differences in memory performances are associated with changes in the expression levels of selected targets involved in neuronal plasticity, energy homeostasis, and stress response. DISCUSSION: Our results suggest that short-term fasting creates an optimal internal state in L. stagnalis' CNS, allowing a spike in insulin release and an upregulation of genes involved in neuroplasticity. Long-term fasting, instead, upregulates genes involved in energy homeostasis and animal survival.

Invertebrates as models of learning and memory: investigating neural and molecular mechanisms.

In this Commentary, we shed light on the use of invertebrates as model organisms for understanding the causal and conserved mechanisms of learning and memory. We provide a condensed chronicle of the contribution offered by mollusks to the studies on how and where the nervous system encodes and stores memory and describe the rich cognitive capabilities of some insect species, including attention and concept learning. We also discuss the use of planarians for investigating the dynamics of memory during brain regeneration and highlight the role of stressful stimuli in forming memories. Furthermore, we focus on the increasing evidence that invertebrates display some forms of emotions, which provides new opportunities for unveiling the neural and molecular mechanisms underlying the complex interaction between stress, emotions and cognition. In doing so, we highlight experimental challenges and suggest future directions that we expect the field to take in the coming years, particularly regarding what we, as humans, need to know for preventing and/or delaying memory loss. This article has an associated ECR Spotlight interview with Veronica Rivi.

No food for thought: an intermediate level of food deprivation enhances memory in Lymnaea stagnalis.

Nutritional status plays an important role in cognitive functioning, but there is disagreement on the role that food deprivation plays in learning and memory. In this study, we investigated the behavioral and transcriptional effects induced by different lengths of food deprivation: 1 day, which is a short time period of food deprivation, and 3 days, which is an 'intermediate' level of food deprivation. Snails were subjected to different feeding regimens and then trained for operant conditioning of aerial respiration, where they received a single 0.5 h training session followed by a long-term memory (LTM) test 24 h later. Immediately after the memory test, snails were killed and the expression levels of key genes for neuroplasticity, energy balance and stress response were measured in the central ring ganglia. We found that 1 day of food deprivation was not sufficient to enhance snails' LTM formation and subsequently did not result in any significant transcriptional effects. However, 3 days of food deprivation resulted in enhanced LTM formation and caused the upregulation of neuroplasticity and stress-related genes and the downregulation of serotonin-related genes. These data provide further insight into how nutritional status and related molecular mechanisms impact cognitive function.

The Role of Dopamine D3 Receptors, Dysbindin, and Their Functional Interaction in the Expression of Key Genes for Neuroplasticity and Neuroinflammation in the Mouse Brain.

Cognitive impairment in schizophrenia remains a clinically and pharmacologically unsolved challenge. Clinical and preclinical studies have revealed that the concomitant reduction in dysbindin (DYS) and dopamine receptor D3 functionality improves cognitive functions. However, the molecular machinery underlying this epistatic interaction has not yet been fully elucidated. The glutamate NMDA receptors and the neurotrophin BDNF, with their established role in promoting neuroplasticity, may be involved in the complex network regulated by the D3/DYS interaction. Furthermore, as inflammation is involved in the etiopathogenesis of several psychiatric diseases, including schizophrenia, the D3/DYS interaction may affect the expression levels of pro-inflammatory cytokines. Thus, by employing mutant mice bearing selective heterozygosis for D3 and/or DYS, we provide new insights into the functional interactions (single and synergic) between these schizophrenia susceptibility genes and the expression levels of key genes for neuroplasticity and neuroinflammation in three key brain areas for schizophrenia: the prefrontal cortex, striatum, and hippocampus. In the hippocampus, the epistatic interaction between D3 and DYS reversed to the wild-type level the downregulated mRNA levels of GRIN1 and GRIN2A were observed in DYS +/- and D3 +/- mice. In all the areas investigated, double mutant mice had higher BDNF levels compared to their single heterozygote counterparts, whereas D3 hypofunction resulted in higher pro-inflammatory cytokines. These results may help to clarify the genetic mechanisms and functional interactions involved in the etiology and development of schizophrenia.

A flavonoid, quercetin, is capable of enhancing long-term memory formation if encountered at different times in the learning, memory formation, and memory recall continuum.

A major extrinsic factor influencing memory and neuro-cognitive performances across taxa is diet. Studies from vertebrates have shown the effects of a flavonoid rich diet on cognitive performance, but the mechanism underlying this action is still poorly understood. A common and abundant flavonoid present in numerous food substances is quercetin (Q). The present study provides the first support for Q-modulated enhancement of cognitive function in an invertebrate model, the pond snail Lymnaea stagnalis, after an operant conditioning procedure. We found that when snails were exposed to Q 3 h before or after a single 0.5 h training session, which typically results in memory lasting ~ 3 h, they formed a long-term memory (LTM) lasting for at least 24 h. Additionally, we assessed the effects of the combined presentation of a single reinforcing stimulus (at 24 h post-training or 24 h before training) and Q-exposure on both LTM formation and reconsolidation. That is, when applied within 3 h of critical periods of memory, Q regulates four different phases: (1) acquisition (i.e., a learning event), (2) consolidation processes after acquisition, (3) memory recall, and (4) memory reconsolidation. In all these phases Q-exposure enhanced LTM persistence.

Non-psychotropic Cannabis sativa L. phytocomplex modulates microglial inflammatory response through CB2 receptors-, endocannabinoids-, and NF-κB-mediated signaling.

Cannabis sativa L. is increasingly emerging for its protective role in modulating neuroinflammation, a complex process orchestrated among others by microglia, the resident immune cells of the central nervous system. Phytocannabinoids, especially cannabidiol (CBD), terpenes, and other constituents trigger several upstream and downstream microglial intracellular pathways. Here, we investigated the molecular mechanisms of a CBD- and terpenes-enriched C. sativa extract (CSE) in an in vitro model of neuroinflammation. We evaluated the effect of CSE on the inflammatory response induced by exposure to lipopolysaccharide (LPS) in BV-2 microglial cells, compared with CBD and ß-caryophyllene (CAR), CB2 receptors (CB2r) inverse and full agonist, respectively. The LPS-induced upregulation of the pro-inflammatory cytokines IL-1ß, IL-6, and TNF-α was significantly attenuated by CSE and only partially by CBD, whereas CAR was ineffective. In BV-2 cells, these anti-inflammatory effects exerted by CSE phytocomplex were only partially dependent on CB2r modulation and they were mediated by the regulation of enzymes responsible for the endocannabinoids metabolism, by the inhibition of reactive oxygen species release and the modulation of JNK/p38 cascade with consequent NF-κB p65 nuclear translocation suppression. Our data suggest that C. sativa phytocomplex and its multitarget mechanism could represent a novel therapeutic strategy for neuroinflammatory-related diseases.

Nature versus nurture in heat stress induced learning between inbred and outbred populations of Lymnaea stagnalis.

Changing environmental conditions often lead to microevolution of traits that are adaptive under the current selection pressure. Currently, one of the major selection pressures is the rise in temperatures globally that has a severe impact on the behavioral ecology of animals. However, the role of thermal stress on neuronal plasticity and memory formation is not well understood. Thermal tolerance and sensitivity to heat stress show variation across populations of the same species experiencing different thermal regimes. We used two populations of the pond snail Lymnaea stagnalis: one lab-bred W-snails and the other wild Delta snails to test heat shock induced learning and memory formation for the Garcia effect learning paradigm. In Garcia effect, a single pairing of a heat stressor (30 °C for 1h) with a novel taste results in a taste-specific negative hedonic shift lasting 24h as long-term memory (LTM) in lab bred W-snails. In this study we used a repeated heat stress procedure to test for increased or decreased sensitivity to the heat before testing for the Garcia effect. We found that lab-bred W-snails show increased sensitivity to heat stress after repeated heat exposure for 7days, leading to enhanced LTM for Garcia effect with only 15min of heat exposure instead of standard 1h. Surprisingly, the freshly collected wild snails do not show Garcia effect. Additionally, F1 generation of wild snails raised and maintained under laboratory conditions still retain their heat stress tolerance similar to their parents and do not show a Garcia effect under standard learning paradigm or even after repeated heat stressor. Thus, we found a differential effect of heat stress on memory formation in wild and lab bred snails. Most interestingly we also show that local environmental (temperature) conditions for one generation is not enough to alter thermal sensitivity in a wild population of L. stagnalis.

Serum metabolic signature of binge-like palatable food consumption in female rats by nuclear magnetic resonance spectroscopy.

Maladaptive eating behavior is a growing public health problem and compulsively eating excessive food in a short time, or binge eating, is a key symptom of many eating disorders. In order to investigate the binge-like eating behavior in female rats, induced by intermittent food restrictions/refeeding and frustration stress, we analyzed for the first time the metabolic profile obtained from serum of rats, through nuclear magnetic resonance (NMR) spectroscopy. In this experimental protocol, rats were exposed to chow food restricting/refeeding and frustration stress manipulation. This stress procedure consists of 15 min exposure to the odor and sight of a familiar chocolate paste, without access to it, just before offering the palatable food. In this model, a "binge-eating episode" was considered the significantly higher palatable food consumption within 2 h in restricted and stressed rats (R + S) than in the other three experimental groups: rats with no food restriction and no stress (NR + NS), only stressed rats (NR + S) or only restricted rats (R + NS). Serum samples from these four different rat groups were collected. The statistical analysis of the 1 H NMR spectral profiles of the four sets of samples pointed to O- and N-acetyl glycoproteins as the main biomarkers for the discrimination of restriction effects. Other metabolites, such as threonine, glycine, glutamine, acetate, pyruvate and lactate, showed trends that may be useful to understand metabolic pathways involved in eating disorders. This study suggested that NMR-based metabolomics is a suitable approach to detect biomarkers related to binge-eating behavior.

To eat or not to eat: a Garcia effect in pond snails (Lymnaea stagnalis).

Taste aversion learning is universal. In animals, a single presentation of a novel food substance followed hours later by visceral illness causes animals to avoid that taste. This is known as bait-shyness or the Garcia effect. Humans demonstrate this by avoiding a certain food following the development of nausea after ingesting that food ('Sauce Bearnaise effect'). Here, we show that the pond snail Lymnaea stagnalis is capable of the Garcia effect. A single 'pairing' of a novel taste, a carrot slurry followed hours later by a heat shock stressor (HS) is sufficient to suppress feeding response elicited by carrot for at least 24 h. Other food tastes are not suppressed. If snails had previously been exposed to carrot as their food source, the Garcia-like effect does not occur when carrot is 'paired' with the HS. The HS up-regulates two heat shock proteins (HSPs), HSP70 and HSP40. Blocking the up-regulation of the HSPs by a flavonoid, quercetin, before the heat shock, prevented the Garcia effect in the snails. Finally, we found that snails exhibit Garcia effect following a period of food deprivation but the long-term memory (LTM) phenotype can be observed only if the animals are tested in a food satiated state.

Long-term memory of configural learning is enhanced via CREB upregulation by the flavonoid quercetin in Lymnaea stagnalis.

Animals respond to acute stressors by modifying their behaviour and physiology. The pond snail Lymnaea stagnalis exhibits configural learning (CL), a form of higher order associative learning. In CL snails develop a landscape of fear when they experience a predatory cue along with a taste of food. This experience results in a suppression of the food response; but the memory only persists for 3 h. Lymnaea has also been found to upregulate heat shock proteins (HSPs) as a result of acute heat stress, which leads to the enhancement of memory formation. A plant flavonoid quercetin blocks the upregulation of HSPs when experienced prior to heat stress. Here, we used this blocking mechanism to test the hypothesis that HSP upregulation plays a critical role in CL. Snails experienced quercetin prior to CL training and surprisingly instead of blocking memory formation it enhanced the memory such that it now persisted for at least 24 h. Quercetin exposure either prior to or after CL enhanced long-term memory (LTM) up to 48 h. We quantified mRNA levels of the transcription factor CREB1 in the Lymnaea central nervous system and found LymCREB1 to be upregulated following quercetin exposure. The enhanced LTM phenotype in L. stagnalis was most pronounced when quercetin was experienced during the consolidation phase. Additionally, quercetin exposure during the memory reconsolidation phase also led to memory enhancement. Thus, we found no support of our original hypothesis but found that quercetin exposure upregulated LymCREB1 leading to LTM formation for CL.

Executive functioning in children with epilepsy: Genes matter.

Pediatric epilepsy has emerged as a chronic medical disease with a characteristic behavioral and cognitive phenotype, which includes compromised executive functioning (EF) and attention-related deficits. However, considerable interindividual variability exists; children often display very different or even opposite outcomes, and some children are more likely than others to develop neurocognitive problems in the face of similar individual and disease-related problems. The factors responsible for this interindividual variability are still largely unknown, but we do know that some genetic factors render the developing brain more susceptible to damage or traumatic experiences than others. Dopamine availability has a neuromodulatory function in the prefrontal cortex (PFC) and especially affects EF. Dopamine availability relates to polymorphisms in the gene encoding catechol-O-methyltransferase (COMT Val158Met), which in turn is affected by the methylation state of its promoter. Allelic variation of the methylenetetrahydrofolate reductase (MTHFR C677T) gene, alters methylation and may influence the methylation state of the COMT promoter. Given this, we tested the hypothesis that these polymorphisms interact in children with epilepsy, and that variability in allelic expression is associated with variability in cognitive phenotype. Executive function was tested directly and indirectly (parent-rated) in 42 children between 5 and 12 years of age. The MTHFR T allele carriers performed worse than MTHFR homozygous CC carriers on indirect EF, and a significant decline was observed when T allele carriers had at least one met allele of the COMT gene, especially on Working Memory. Direct EF was significantly compromised in COMT Val/Val carriers where reduced dopamine availability seems to confer a higher risk in a test that requests a high degree of executive attention and planning. This finding suggests that in children with epilepsy, genes that influence methylation and dopamine availability affect PFC-related EF. Therefore, we should consider genetic vulnerability as a polygenic risk, which might predispose for a particular phenotype and include specific genetic signatures as part of each patient's behavioral and cognitive profile from the moment that we start to take care of the child.

LPS-induced histone H3 phospho(Ser10)-acetylation(Lys14) regulates neuronal and microglial neuroinflammatory response.

Epigenetic modifications of DNA and histone proteins are emerging as fundamental mechanisms by which neural cells adapt their transcriptional response to environmental cues, such as, immune stimuli or stress. In particular, histone H3 phospho(Ser10)-acetylation(Lys14) (H3S10phK14ac) has been linked to activation of specific gene expression. The purpose of this study was to investigate the role of H3S10phK14ac in a neuroinflammatory condition. Adult male rats received a intraperitoneal injection of lipopolysaccharide (LPS) (830 µg/Kg/i.p., n = 6) or vehicle (saline 1 mL/kg/i.p., n = 6) and were sacrificed 2 or 6 h later. We showed marked region- and time-specific increases in H3S10phK14ac in the hypothalamus and hippocampus, two principal target regions of LPS. These changes were accompanied by a marked transcriptional activation of interleukin (IL) 1ß, IL-6, Tumour Necrosis Factor (TNF) α, the inducible nitric oxide synthase (iNOS) and the immediate early gene c-Fos. By means of chromatin immunoprecipitation, we demonstrated an increased region- and time-specific association of H3S10phK14ac with the promoters of IL-6, c-Fos and iNOS genes, suggesting that part of the LPS-induced transcriptional activation of these genes is regulated by H3S10phK14ac. Finally, by means of multiple immunofluorescence approach, we showed that increased H3S10phK14ac is cell type-specific, being neurons and reactive microglia, the principal histological types involved in this response. Present data point to H3S10phK14ac as a principal epigenetic regulator of neural cell response to systemic LPS and underline the importance of distinct time-, region- and cell-specific epigenetic mechanisms that regulate gene transcription to understand the mechanistic complexity of neuroinflammatory response to immune challenges.

The Proinflammatory Cytokine Interleukin 18 Regulates Feeding by Acting on the Bed Nucleus of the Stria Terminalis.

UNLABELLED: The proinflammatory cytokine IL-18 has central anorexigenic effects and was proposed to contribute to loss of appetite observed during sickness. Here we tested in the mouse the hypothesis that IL-18 can decrease food intake by acting on neurons of the bed nucleus of the stria terminalis (BST), a component of extended amygdala recently shown to influence feeding via its projections to the lateral hypothalamus (LH). We found that both subunits of the heterodimeric IL-18 receptor are highly expressed in the BST and that local injection of recombinant IL-18 (50 ng/ml) significantly reduced c-fos activation and food intake for at least 6 h. Electrophysiological experiments performed in BST brain slices demonstrated that IL-18 strongly reduces the excitatory input on BST neurons through a presynaptic mechanism. The effects of IL-18 are cell-specific and were observed in Type III but not in Type I/II neurons. Interestingly, IL-18-sensitve Type III neurons were recorded in the juxtacapsular BST, a region that contains BST-LH projecting neurons. Reducing the excitatory input on Type III GABAergic neurons, IL-18 can increase the firing of glutamatergic LH neurons through a disinhibitory mechanism. Imbalance between excitatory and inhibitory activity in the LH can induce changes in food intake. Effects of IL-18 were mediated by the IL-18R because they were absent in neurons from animals null for IL-18Rα (Il18ra(-/-)), which lack functional IL-18 receptors. In conclusion, our data show that IL-18 may inhibit feeding by inhibiting the activity of BST Type III GABAergic neurons. SIGNIFICANCE STATEMENT: Loss of appetite during sickness is a common and often debilitating phenomenon. Although proinflammatory cytokines are recognized as mediators of these anorexigenic effects, their mechanism and sites of action remain poorly understood. Here we show that interleukin 18, an anorexigenic cytokine, can act on neurons of the bed nucleus of the stria terminalis to reduce food intake via the IL-18 receptor. The findings identify a site and a mode of action that indicate targets for the treatment of cachexia or other eating disorders.

Interleukin 18 activates MAPKs and STAT3 but not NF-κB in hippocampal HT-22 cells.

Interleukin (IL)-18 is a cytokine previously demonstrated to participate in neuroinflammatory processes. Since the components of the IL-18 receptor complex are expressed in neurons throughout the brain, IL-18 is also believed to directly influence neuronal function. Here we tested this hypothesis on mouse hippocampal neurons by measuring the effects of IL-18 on three pathways previously shown to be regulated by this cytokine in non-neuronal cells: the MAPK pathways, p38 and ERK1/2 MAPKs, STAT3 and NF-κB. Experiments were carried out in vitro using the immortalized hippocampal neuronal line HT-22 or in vivo following i.c.v. injection with recombinant mouse IL-18. We showed that IL-18 did not activate NF-κB in HT-22 cells whereas it induced a rapid (within 15min) activation of the MAPK pathways. Moreover, we demonstrated that IL-18 treatment enhanced P-STAT3 (Tyr705)/STAT3 ratio in the nucleus of HT-22 cells after 30-60min of exposure. A similar increase in P-STAT3 (Tyr705)/STAT3 ratio was observed in the whole hippocampus one hour after i.c.v. injection. These data demonstrate that IL-18 can act directly on neuronal cells affecting the STAT3 pathway; therefore, possibly regulating the expression of specific genes within the hippocampus. This effect may help to explain some of the IL-18-induced effects on synaptic plasticity and functionality within the hippocampal system.

Snails go on a fast when acetylsalicylic acid comes along with heat stress: A possible effect of HSPs and serotonergic system on the feeding response.

A novel food followed by sickness, causes a taste-specific conditioned aversion, known as the 'Garcia effect'. We recently found that both a heat shock stressor (30 °C for 1 h - HS) and the bacterial lipopolysaccharide (LPS) can be used as 'sickness-inducing' stimuli to induce a Garcia effect in the pond snail Lymnaea stagnalis. Additionally, if snails are exposed to acetylsalicylic acid (ASA) present in aspirin tablets before the LPS injection, the formation of the Garcia effect is prevented. Here, we hypothesized that exposing snails to crushed aspirin before the HS (ASA-HS) would prevent the HS-induced 'sickness state' and - therefore -the Garcia effect. Unexpectantly, the ASA-HS procedure induced a generalized and long-lasting feeding suppression. We thus investigate the molecular effects underlying this phenomenon. While the exposure to the HS alone resulted in a significant upregulation of the mRNA levels of the Heat Shock Protein 70 (HSP 70) in snails' central ring ganglia, the ASA-HS procedure induced an even greater upregulation of HSP70, suggesting that the ASA-HS combination causes a severe stress response that inhibits feeding. Additionally, we found that the ASA-HS procedure induced a significant downregulation of the mRNA levels of genes involved with the serotoninergic system which regulates feeding in snails. Finally, the ASA-HS procedure prevented HS-induced upregulation of the mRNA levels of key neuroplasticity genes. Our study indicates that two sickness-inducing stimuli can have different physiological responses even if behavioral outcomes are similar under some learning contexts.