Nucleotides as Bacterial Second Messengers.

In addition to comprising monomers of nucleic acids, nucleotides have signaling functions and act as second messengers in both prokaryotic and eukaryotic cells. The most common example is cyclic AMP (cAMP). Nucleotide signaling is a focus of great interest in bacteria. Cyclic di-AMP (c-di-AMP), cAMP, and cyclic di-GMP (c-di-GMP) participate in biological events such as bacterial growth, biofilm formation, sporulation, cell differentiation, motility, and virulence. Moreover, the cyclic-di-nucleotides (c-di-nucleotides) produced in pathogenic intracellular bacteria can affect eukaryotic host cells to allow for infection. On the other hand, non-cyclic nucleotide molecules pppGpp and ppGpp are alarmones involved in regulating the bacterial response to nutritional stress; they are also considered second messengers. These second messengers can potentially be used as therapeutic agents because of their immunological functions on eukaryotic cells. In this review, the role of c-di-nucleotides and cAMP as second messengers in different bacterial processes is addressed.

Cyclic di-AMP traps proton-coupled K+ transporters of the KUP family in an inward-occluded conformation.

Cyclic di-AMP is the only known essential second messenger in bacteria and archaea, regulating different proteins indispensable for numerous physiological processes. In particular, it controls various potassium and osmolyte transporters involved in osmoregulation. In Bacillus subtilis, the K+/H+ symporter KimA of the KUP family is inactivated by c-di-AMP. KimA sustains survival at potassium limitation at low external pH by mediating potassium ion uptake. However, at elevated intracellular K+ concentrations, further K+ accumulation would be toxic. In this study, we reveal the molecular basis of how c-di-AMP binding inhibits KimA. We report cryo-EM structures of KimA with bound c-di-AMP in detergent solution and reconstituted in amphipols. By combining structural data with functional assays and molecular dynamics simulations we reveal how c-di-AMP modulates transport. We show that an intracellular loop in the transmembrane domain interacts with c-di-AMP bound to the adjacent cytosolic domain. This reduces the mobility of transmembrane helices at the cytosolic side of the K+ binding site and therefore traps KimA in an inward-occluded conformation.

Cyclic-di-AMP signalling in lactic acid bacteria.

Cyclic dimeric adenosine monophosphate (cyclic-di-AMP) is a nucleotide second messenger present in Gram-positive bacteria, Gram-negative bacteria and some Archaea. The intracellular concentration of cyclic-di-AMP is adjusted in response to environmental and cellular cues, primarily through the activities of synthesis and degradation enzymes. It performs its role by binding to protein and riboswitch receptors, many of which contribute to osmoregulation. Imbalances in cyclic-di-AMP can lead to pleiotropic phenotypes, affecting aspects such as growth, biofilm formation, virulence, and resistance to osmotic, acid, and antibiotic stressors. This review focuses on cyclic-di-AMP signalling in lactic acid bacteria (LAB) incorporating recent experimental discoveries and presenting a genomic analysis of signalling components from a variety of LAB, including those found in food, and commensal, probiotic, and pathogenic species. All LAB possess enzymes for the synthesis and degradation of cyclic-di-AMP, but are highly variable with regards to the receptors they possess. Studies in Lactococcus and Streptococcus have revealed a conserved function for cyclic-di-AMP in inhibiting the transport of potassium and glycine betaine, either through direct binding to transporters or to a transcriptional regulator. Structural analysis of several cyclic-di-AMP receptors from LAB has also provided insights into how this nucleotide exerts its influence.

Regulation of cardiac function by cAMP nanodomains.

Cyclic adenosine monophosphate (cAMP) is a diffusible intracellular second messenger that plays a key role in the regulation of cardiac function. In response to the release of catecholamines from sympathetic terminals, cAMP modulates heart rate and the strength of contraction and ease of relaxation of each heartbeat. At the same time, cAMP is involved in the response to a multitude of other hormones and neurotransmitters. A sophisticated network of regulatory mechanisms controls the temporal and spatial propagation of cAMP, resulting in the generation of signaling nanodomains that enable the second messenger to match each extracellular stimulus with the appropriate cellular response. Multiple proteins contribute to this spatiotemporal regulation, including the cAMP-hydrolyzing phosphodiesterases (PDEs). By breaking down cAMP to a different extent at different locations, these enzymes generate subcellular cAMP gradients. As a result, only a subset of the downstream effectors is activated and a specific response is executed. Dysregulation of cAMP compartmentalization has been observed in cardiovascular diseases, highlighting the importance of appropriate control of local cAMP signaling. Current research is unveiling the molecular organization underpinning cAMP compartmentalization, providing original insight into the physiology of cardiac myocytes and the alteration associated with disease, with the potential to uncover novel therapeutic targets. Here, we present an overview of the mechanisms that are currently understood to be involved in generating cAMP nanodomains and we highlight the questions that remain to be answered.

Measuring Spatiotemporal cAMP Dynamics Within an Endogenous Signaling Compartment Using FluoSTEP-ICUE.

cAMP is a ubiquitous second messenger involved in the regulation of diverse cellular processes. Spatiotemporal regulation of cAMP through compartmentalization within various subcellular microdomains is essential to ensure specific signaling. In the following protocol, we describe a method for directly visualizing signaling dynamics within cAMP microdomains using fluorescent sensors targeted to endogenous proteins (FluoSTEPs). Instead of overexpressing a biosensor-tagged protein of interest to target a microdomain, FluoSTEP Indicator of cAMP using Epac (FluoSTEP-ICUE) utilizes spontaneously complementing split GFP and CRISPR-Cas9 genome editing to localize a FRET-based cAMP biosensor to an endogenously expressed protein of interest. Utilizing this approach, FluoSTEP-ICUE can be used to measure cAMP levels within endogenous signaling compartments, thus providing a powerful tool for studying the spatiotemporal regulation of cAMP signaling.

Phosphodiesterases and Compartmentation of cAMP and cGMP Signaling in Regulation of Cardiac Contractility in Normal and Failing Hearts.

Cardiac contractility is regulated by several neural, hormonal, paracrine, and autocrine factors. Amongst these, signaling through ß-adrenergic and serotonin receptors generates the second messenger cyclic AMP (cAMP), whereas activation of natriuretic peptide receptors and soluble guanylyl cyclases generates cyclic GMP (cGMP). Both cyclic nucleotides regulate cardiac contractility through several mechanisms. Phosphodiesterases (PDEs) are enzymes that degrade cAMP and cGMP and therefore determine the dynamics of their downstream effects. In addition, the intracellular localization of the different PDEs may contribute to regulation of compartmented signaling of cAMP and cGMP. In this review, we will focus on the role of PDEs in regulating contractility and evaluate changes in heart failure.

The c-di-AMP signaling system influences stress tolerance and biofilm formation of Streptococcus mitis.

Streptococcus mitis is a commensal bacterial species of the oral cavity, with the potential for opportunistic pathogenesis. For successful colonization, S. mitis must be able to adhere to surfaces of the oral cavity and survive and adapt to frequently changing environmental conditions. Cyclic-di-AMP (c-di-AMP) is a nucleotide second messenger, involved in the regulation of stress responses and biofilm formation in several bacterial species. Cyclic-di-AMP is produced by diadenylate cyclases and degraded by phosphodiesterases. We have previously shown that in S. mitis, one diadenylate cyclase (CdaA) and at least two phosphodiesterases (Pde1 and Pde2) regulate the intracellular concentration of c-di-AMP. In this study, we utilized S. mitis deletion mutants of cdaA, pde1, and pde2 to analyze the role of c-di-AMP signaling in various stress responses, biofilm formation, and adhesion to eukaryotic cells. Here, we demonstrate that the Δpde1 mutant displayed a tendency toward increased susceptibility to acetic acid at pH 4.0. Deletion of cdaA increases auto-aggregation of S. mitis but reduces biofilm formation on an abiotic surface. These phenotypes are more pronounced under acidic extracellular conditions. Inactivation of pde1 or pde2 reduced the tolerance to ciprofloxacin, and UV radiation and the Δpde1 mutant was more susceptible to Triton X-100, indicating a role for c-di-AMP signaling in responses to DNA damage and cell membrane perturbation. Finally, the Δpde2 mutant displayed a tendency toward a reduced ability to adhere to oral keratinocytes. Taken together, our results indicate an important role for c-di-AMP signaling in cellular processes important for colonization of the mouth.

Endoplasmic Reticulum-Plasma Membrane Contact Sites as an Organizing Principle for Compartmentalized Calcium and cAMP Signaling.

In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER-PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.

Role of protons in calcium signaling.

Thirty-six years after the publication of the important article by Busa and Nuccitelli on the variability of intracellular pH (pHi) and the interdependence of pHi and intracellular Ca2+ concentration ([Ca2+]i), little research has been carried out on pHi and calcium signaling. Moreover, the results appear to be contradictory. Some authors claim that the increase in [Ca2+]i is due to a reduction in pHi, others that it is caused by an increase in pHi. The reasons for these conflicting results have not yet been discussed and clarified in an exhaustive manner. The idea that variations in pHi are insignificant, because cellular buffers quickly stabilize the pHi, may be a limiting and fundamentally wrong concept. In fact, it has been shown that protons can move and react in the cell before they are neutralized. Variations in pHi have a remarkable impact on [Ca2+]i and hence on some of the basic biochemical mechanisms of calcium signaling. This paper focuses on the possible triggering role of protons during their short cellular cycle and it suggests a new hypothesis for an IP3 proton dependent mechanism of action.

Thrombospondin-1 promotes hemostasis through modulation of cAMP signaling in blood platelets.

Thrombospondin-1 (TSP-1) is released by platelets upon activation and can increase platelet activation, but its role in hemostasis in vivo is unclear. We show that TSP-1 is a critical mediator of hemostasis that promotes platelet activation by modulating inhibitory cyclic adenosine monophosphate (cAMP) signaling. Genetic deletion of TSP-1 did not affect platelet activation in vitro, but in vivo models of hemostasis and thrombosis showed that TSP-1-deficient mice had prolonged bleeding, defective thrombosis, and increased sensitivity to the prostacyclin mimetic iloprost. Adoptive transfer of wild-type (WT) but not TSP-1-/- platelets ameliorated the thrombotic phenotype, suggesting a key role for platelet-derived TSP-1. In functional assays, TSP-1-deficient platelets showed an increased sensitivity to cAMP signaling, inhibition of platelet aggregation, and arrest under flow by prostacyclin (PGI2). Plasma swap experiments showed that plasma TSP-1 did not correct PGI2 hypersensitivity in TSP-1-/- platelets. By contrast, incubation of TSP-1-/- platelets with releasates from WT platelets or purified TSP-1, but not releasates from TSP-1-/- platelets, reduced the inhibitory effects of PGI2. Activation of WT platelets resulted in diminished cAMP accumulation and downstream signaling, which was associated with increased activity of the cAMP hydrolyzing enzyme phosphodiesterase 3A (PDE3A). PDE3A activity and cAMP accumulation were unaffected in platelets from TSP-1-/- mice. Platelets deficient in CD36, a TSP-1 receptor, showed increased sensitivity to PGI2/cAMP signaling and diminished PDE3A activity, which was unaffected by platelet-derived or purified TSP-1. This scenario suggests that the release of TSP-1 regulates hemostasis in vivo through modulation of platelet cAMP signaling at sites of vascular injury.

Sustained Control of Pyruvate Carboxylase by the Essential Second Messenger Cyclic di-AMP in Bacillus subtilis.

In Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. In some bacteria, c-di-AMP also binds to the pyruvate carboxylase to inhibit its activity. We have discovered that in B. subtilis the c-di-AMP target protein DarB, rather than c-di-AMP itself, specifically binds to pyruvate carboxylase both in vivo and in vitro. This interaction stimulates the activity of the enzyme, as demonstrated by in vitro enzyme assays and in vivo metabolite determinations. Both the interaction and the activation of enzyme activity require apo-DarB and are inhibited by c-di-AMP. Under conditions of potassium starvation and corresponding low c-di-AMP levels, the demand for citric acid cycle intermediates is increased. Apo-DarB helps to replenish the cycle by activating both pyruvate carboxylase gene expression and enzymatic activity via triggering the stringent response as a result of its interaction with the (p)ppGpp synthetase Rel and by direct interaction with the enzyme, respectively. IMPORTANCE If bacteria experience a starvation for potassium, by far the most abundant metal ion in every living cell, they have to activate high-affinity potassium transporters, switch off growth activities such as translation and transcription of many genes or replication, and redirect the metabolism in a way that the most essential functions of potassium can be taken over by metabolites. Importantly, potassium starvation triggers a need for glutamate-derived amino acids. In many bacteria, the responses to changing potassium availability are orchestrated by a nucleotide second messenger, cyclic di-AMP. c-di-AMP binds to factors involved directly in potassium homeostasis and to dedicated signal transduction proteins. Here, we demonstrate that in the Gram-positive model organism Bacillus subtilis, the c-di-AMP receptor protein DarB can bind to and, thus, activate pyruvate carboxylase, the enzyme responsible for replenishing the citric acid cycle. This interaction takes place under conditions of potassium starvation if DarB is present in the apo form and the cells are in need of glutamate. Thus, DarB links potassium availability to the control of central metabolism.

Adenylate Cyclase Toxin Tinkering With Monocyte-Macrophage Differentiation.

Circulating inflammatory monocytes are attracted to infected mucosa and differentiate into macrophage or dendritic cells endowed with enhanced bactericidal and antigen presenting capacities. In this brief Perspective we discuss the newly emerging insight into how the cAMP signaling capacity of Bordetella pertussis adenylate cyclase toxin manipulates the differentiation of monocytes and trigger dedifferentiation of the alveolar macrophages to facilitate bacterial colonization of human airways.

The second messenger c-di-AMP mediates bacterial exopolysaccharide biosynthesis: a review.

Cyclic dimeric adenosine 3'-5'-monophosphate (c-di-AMP) is a recently discovered nucleotide messenger in bacteria. It plays an important role in signaling, transcription, and cell physiology, such as in bacterial growth, potassium transport, fatty acid synthesis, the metabolic balance of cell wall components, and biofilm formation. Exopolysaccharides (EPSs) have distinct physico-chemical properties and diverse bioactivities including antibacterial, hypolipidemic, and antioxidative activities, and they are widely used in the food, pharmaceutical, and cosmetic industries. Although c-di-AMP has been demonstrated to regulate the biosynthesis of bacterial EPSs, only a single c-di-AMP receptor, CabpA, has been identified in EPS synthesis. With the aim of describing current understanding of the regulation of microbial EPSs, this review summarizes c-di-AMP biosynthesis and degradation as well as the mechanism through which c-di-AMP regulates bacterial EPSs.

The Many Roles of the Bacterial Second Messenger Cyclic di-AMP in Adapting to Stress Cues.

Bacteria respond to changes in environmental conditions through adaptation to external cues. Frequently, bacteria employ nucleotide signaling molecules to mediate a specific, rapid response. Cyclic di-AMP (c-di-AMP) was recently discovered to be a bacterial second messenger that is essential for viability in many species. In this review, we highlight recent work that has described the roles of c-di-AMP in bacterial responses to various stress conditions. These studies show that depending on the lifestyle and environmental niche of the bacterial species, the c-di-AMP signaling network results in diverse outcomes, such as regulating osmolyte transport, controlling plant attachment, or providing a checkpoint for spore formation. c-di-AMP achieves this signaling specificity through expression of different classes of synthesis and catabolic enzymes as well as receptor proteins and RNAs, which will be summarized.

Metabolites released from apoptotic cells act as tissue messengers.

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2-4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues.

Distinct temporal integration of noradrenaline signaling by astrocytic second messengers during vigilance.

Astrocytes may function as mediators of the impact of noradrenaline on neuronal function. Activation of glial α1-adrenergic receptors triggers rapid astrocytic Ca2+ elevation and facilitates synaptic plasticity, while activation of ß-adrenergic receptors elevates cAMP levels and modulates memory consolidation. However, the dynamics of these processes in behaving mice remain unexplored, as do the interactions between the distinct second messenger pathways. Here we simultaneously monitored astrocytic Ca2+ and cAMP and demonstrate that astrocytic second messengers are regulated in a temporally distinct manner. In behaving mice, we found that while an abrupt facial air puff triggered transient increases in noradrenaline release and large cytosolic astrocytic Ca2+ elevations, cAMP changes were not detectable. By contrast, repeated aversive stimuli that lead to prolonged periods of vigilance were accompanied by robust noradrenergic axonal activity and gradual sustained cAMP increases. Our findings suggest distinct astrocytic signaling pathways can integrate noradrenergic activity during vigilance states to mediate distinct functions supporting memory.

Bacterial Second Messenger Cyclic di-AMP Modulates the Competence State in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is a naturally competent organism that causes diseases such as pneumonia, otitis media, and bacteremia. The essential bacterial second messenger cyclic di-AMP (c-di-AMP) is an emerging player in the stress responses of many pathogens. In S. pneumoniae, c-di-AMP is produced by a diadenylate cyclase, CdaA, and cleaved by phosphodiesterases Pde1 and Pde2. c-di-AMP binds a transporter of K+ (Trk) family protein, CabP, which subsequently halts K+ uptake via the transporter TrkH. Recently, it was reported that Pde1 and Pde2 are essential for pneumococcal virulence in mouse models of disease. To elucidate c-di-AMP-mediated transcription that may lead to changes in pathogenesis, we compared the transcriptomes of wild-type (WT) and Δpde1 Δpde2 strains by transcriptome sequencing (RNA-Seq) analysis. Notably, we found that many competence-associated genes are significantly upregulated in the Δpde1 Δpde2 strain compared to the WT. These genes play a role in DNA uptake, recombination, and autolysis. Competence is induced by a quorum-sensing mechanism initiated by the secreted factor competence-stimulating peptide (CSP). Surprisingly, the Δpde1 Δpde2 strain exhibited reduced transformation efficiency compared to WT bacteria, which was c-di-AMP dependent. Transformation efficiency was also directly related to the [K+] in the medium, suggesting a link between c-di-AMP function and the pneumococcal competence state. We found that a strain that possesses a V76G variation in CdaA produced less c-di-AMP and was highly susceptible to CSP. Deletion of cabP and trkH restored the growth of these bacteria in medium with CSP. Overall, our study demonstrates a novel role for c-di-AMP in the competence program of S. pneumoniaeIMPORTANCE Genetic competence in bacteria leads to horizontal gene transfer, which can ultimately affect antibiotic resistance, adaptation to stress conditions, and virulence. While the mechanisms of pneumococcal competence signaling cascades have been well characterized, the molecular mechanism behind competence regulation is not fully understood. The bacterial second messenger c-di-AMP has previously been shown to play a role in bacterial physiology and pathogenesis. In this study, we provide compelling evidence for the interplay between c-di-AMP and the pneumococcal competence state. These findings not only attribute a new biological function to this dinucleotide as a regulator of competence, transformation, and survival under stress conditions in pneumococci but also provide new insights into how pneumococcal competence is modulated.

Lens Connexin Channels Have Differential Permeability to the Second Messenger cAMP.

Purpose: Gap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation. Methods: We compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule. Results: cAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46. Conclusions: These data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.

Nucleotide second messengers in Streptomyces.

Antibiotic producing Streptomyces sense and respond to environmental signals by using nucleotide second messengers, including (p)ppGpp, cAMP, c-di-GMP and c-di-AMP. As summarized in this review, these molecules are important message carriers that coordinate the complex Streptomyces morphological transition from filamentous growth to sporulation along with the secondary metabolite production. Here, we provide an overview of the enzymes that make and break these second messengers and suggest candidates for (p)ppGpp and cAMP enzymes to be studied. We highlight the target molecules that bind these signalling molecules and elaborate individual functions that they control in the context of Streptomyces development. Finally, we discuss open questions in the field, which may guide future studies in this exciting research area.

Geometric principles of second messenger dynamics in dendritic spines.

Dendritic spines are small, bulbous protrusions along dendrites in neurons and play a critical role in synaptic transmission. Dendritic spines come in a variety of shapes that depend on their developmental state. Additionally, roughly 14-19% of mature spines have a specialized endoplasmic reticulum called the spine apparatus. How does the shape of a postsynaptic spine and its internal organization affect the spatio-temporal dynamics of short timescale signaling? Answers to this question are central to our understanding the initiation of synaptic transmission, learning, and memory formation. In this work, we investigated the effect of spine and spine apparatus size and shape on the spatio-temporal dynamics of second messengers using mathematical modeling using reaction-diffusion equations in idealized geometries (ellipsoids, spheres, and mushroom-shaped). Our analyses and simulations showed that in the short timescale, spine size and shape coupled with the spine apparatus geometries govern the spatiotemporal dynamics of second messengers. We show that the curvature of the geometries gives rise to pseudo-harmonic functions, which predict the locations of maximum and minimum concentrations along the spine head. Furthermore, we showed that the lifetime of the concentration gradient can be fine-tuned by localization of fluxes on the spine head and varying the relative curvatures and distances between the spine apparatus and the spine head. Thus, we have identified several key geometric determinants of how the spine head and spine apparatus may regulate the short timescale chemical dynamics of small molecules that control synaptic plasticity.