extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
The Present and the Future of Degradable Dendrimers and Derivatives in Theranostics

Interest in dendrimer-based nanomedicines has been growing recently, as it is possible to precisely manipulate the molecular weight, chemical composition, and surface functionality of dendrimers, tuning their properties according to the desired biomedical application. However, one important concern about dendrimer-based therapeutics remains-the nondegradability under physiological conditions of the most commonly used dendrimers. Therefore, biodegradable dendrimers represent an attractive class of nanomaterials, since they present advantages over conventional nondegradable dendrimers regarding the release of the loaded molecules and the prevention of bioaccumulation of synthetic materials and subsequent cytotoxicity. Here, we present an overview of the state-of-the-art of the design of biodegradable dendritic structures, with particular focus on the hurdles regarding the use of these as vectors of drugs and nucleic acids, as well as macromolecular contrast agents.

INTRODUCTION
Natural and synthetic macromolecular structures have frequently been used as vectors for the delivery of drugs and therapeutic nucleic acids, as well as diagnostic agents. This concept emerged from the hypothesis that these could protect the molecule of interest from undesirable interactions with components of the biological milieu along with improving its solubility. Several carriers have been studied: linear polymers, micellar assemblies, liposomes, polymersomes, and, more recently, dendritic structures (dendrimers and dendrons).(1) The ideal carrier should facilitate high drug loading, long blood circulation time (in the case of the commonly explored intravenous administration), high accumulation in the desired tissue, low toxicity, low immunogenicity, simplicity in its preparation, and, preferably, adequate biodegradability.
Dendritic structures emerged from a new class of highly branched polymers, first synthesized by Voegtle and colleagues in 1978, and coined "cascade molecules".(2) Later on, Denkewalter, Tomalia, Newkome, Frechet, and co-workers further increased the level of complexity of these branched molecules, giving rise to larger dendritic structures that were then renamed "dendrimers".(3) Dendrimers consist of the following: (a) a central core with two or more reactive groups, (b) repeated units or monomers covalently attached to the central core and organized in layers called "generations" (G), and (c) terminal functional groups on their surface. They can be synthesized by two different approaches: divergent(3b) or convergent.(3d) The most commonly used dendrimers are poly(amido amine) (PAMAM),(3b) poly(propylene imine) (PPI),(2) and poly(lysine)-based dendrimers;(4) currently, all are commercially available. These and other dendrimers have been proposed as promising carriers for drug delivery(5) due to their unique structural characteristics: globular, well-defined, and very branched structure, as well as their monodispersion and controllable nanosize. Moreover, the presence of a high density of terminal functional groups allows the tethering of different ligands and/or drugs in a specific and controllable manner, simulating the multivalency present in different biological systems. This multivalency is the greater virtue of dendrimers: the enhanced effect that stems from presenting lots of several bioactive molecules at the same time and place. Additionally, dendrimers can also cargo a molecule of interest by forming nanosized structures stabilized by noncovalent interactions.
The characteristics of the surface groups of the dendrimers, besides determining predominantly their physicochemical properties, will also determine their biological activity and biocompatibility. For example, cationic dendrimers will more readily interact with the negatively charged surface of the cell membranes, but have also been found to have more cytotoxicity than anionic or neutral ones.(6) A common approach for masking the surface charge and, in general, improving the biocompatibility of dendrimers and/or dendrons, as well as increasing their circulation time in the bloodstream, is to tether to the dendrimers backbone chains of poly(ethylene glycol) (PEG).(7) This strategy was also carried out in the case of DEP docetaxel, a dendrimer with docetaxel attached that is in Phase 1 clinical trials for the treatment of a wide range of solid tumors including breast, lung, and prostate. Other dendrimers have also reached clinical trials: VivaGel, a G4 poly(l-lysine)-based dendrimer, which acts as an antimicrobial agent being applied in the treatment/prevention of a range of sexually transmitted diseases, is in Phase 3 trials; and Gadomer-17, a poly(lysine)-based dendrimer bearing 24 Gd(III)-DOTA chelates (commercial name Dotarem; DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), is in Phase 2 trials for its use as a dendritic contrast agent for diagnosis in magnetic resonance imaging (MRI).
Despite the progress in the design of dendritic structures with improved features for biomedical application, one of the main drawbacks of the most currently used dendrimers is their nondegradability under physiological conditions that can result in cytotoxicity induced by the accumulation of nondegradable synthetic materials inside cells or in tissues. (8) In vitro studies have shown that dendrimer cytotoxicity is mainly associated with cell membrane disruption and subsequent necrosis/non-apoptotic cell death.(6a, 8d) Numerous works have thoroughly described the effect of dendrimer chemistry, size, and charge on biological membrane integrity. (8c, 9) However, recent reports suggest that, apart from membrane destabilization, toxicity may also arise from impaired oxidative metabolism resulting from mitochondrial dysfunction(10) and changes in endogenous gene expression(11) that ultimately lead to apoptotic cell death.(10, 11) Dendrimer chemistry, charge, and size are features that will also have an impact on in vivo biodistribution and pharmacokinetics.(6a, 8c, 9a) Additionally, in vivo cytotoxicity will also depend on the dose and the administration route.(6a) However, conflicting data can be found in the open literature concerning in vivo testing. Some studies report that PAMAM and PPI dendrimers, especially at low generations, are not as toxic as initially described.(12) However, others have reported toxicity profiles for the same dendrimers. (13) Consequently, a number of teams has been focusing on the design of biodegradable dendritic structures, to overcome these hurdles. The use of biodegradable materials that, under biological environmental conditions, degrade in time into smaller fragments that can be excreted or eliminated through metabolic pathways is expected to overcome the risk of complications associated with the long-term presence of high-molecular-weight compounds.
The development of biodegradable dendritic structures has also been put forward in the context of the design of "smart" controlled delivery systems in which one aims at triggering and/or sustaining the release of a therapeutic agent via the control of the degradation profile of its vector. Inclusively, some authors have proposed the use of degradable dendritic structures as contrasting agents that can overcome long-term Gd(III) tissue accumulation. (14) Here, we present the state-of-the-art of biodegradable dendritic structures, with particular focus on the ones used in the context of drug and nucleic acid delivery, as well as their use as macromolecular diagnostic agents. The design and synthesis of such structures are presented, as well as the challenges that remain ahead toward their widespread application. Starting from the assumption that at least some animal research is relevant, ethically acceptable and presently not replaceable, some harm to animals in research may be perceived as a 'necessary evil', in particular in face of the moral importance of advancing biomedical knowledge for the benefit of humans and non-humans alike. However, it should nevertheless be reflected upon in which circumstances it may -or may not -be either 'necessary' or 'evil' to kill animals in the context of animal research. In this chapter, we discuss whether killing is inevitable, or morally problematic, as well as to whom this killing matters.

GETTING AND TUNING DEGRADABILITY
As in the case of linear polymers, the biodegradability of dendrimers can be achieved by inclusion in their structure of labile bonds to be broken due to a specific biological activity or stimulus. To the best of our knowledge, the majority of efforts have been focused on the development of dendritic architectures (dendrimers, dendrons, and linear-dendritic block copolymers) with hydrolyzable bonds. The functionalities more susceptible to hydrolytic cleavage are based on anhydrides, esters, phosphoesters, carbamates, ethers, and amides. The main factors that control the rate of degradation of dendritic structures are (a) the chemical bond present on or connecting the monomer units; (b) the hydrophobicity of the monomer units-more hydrophilic monomers result in faster degrading structures compared to hydrophobic monomers; (c) size-larger dendrimers degrade more slowly compared to smaller ones due to the higher packaging; and (d) the localization of the cleavable linkages, since the hydrolysis of interior linkages leads to faster degradation of the whole dendrimer. With these parameters taken into account, it is expected that one can modulate the properties of biodegradable dendritic structures to achieve the desired degradation rate for a specific application.
The covalent degradation or dendrimer fragmentation could proceed through removal of a certain percentage of surface functionalities, removal of dendritic branches or monomers, or removal of the central core. Therefore, the cleavage of only certain parts of the backbone of the designed dendrimer could lead to its full degradation into low-molecular-weight fragments. Moreover, it is worthwhile mentioning that the degradation of some cleavage polymeric materials (like aliphatic polyesters) could also be accelerated by the presence of superoxide ions (O2-) generated in the body fluids and tissues during inflammatory response to foreign materials.(15)

BIODEGRADABLE DENDRITIC STRUCTURES
The different types of biodegradable dendritic structures can be classified according to the type of degradable connection or bond, the monomer on which they are based, and the synthetic strategy to prepare them, among others.
Although very few examples of biodegradable dendritic structures not based on ester bonds can be found (see section 3.4), the majority of the dendrimers that are susceptible to degradation under physiological conditions reported so far contain ester bonds as focal points of degradation. Polyester dendrimers represent an attractive class of nanomaterials due to their good biocompatibility and the compromise between their biodegradability trait and the possibility of synthetic manipulation in comparison with other more hydrolytic susceptible structures, such as polyanhydrides. Even so, the synthesis of these nanocarriers is challenging because of the hydrolytic susceptibility of the ester bond.(16) In contrast, polyethers and polyamide-based dendrimers could withstand much wider selection of synthetic manipulations, yet they do not degrade easily in the body but only under severe conditions of hydrolysis. Thus, these dendritic structures may be more prone to long-term accumulation in vivo. Therefore, these types of dendrimers will not be considered in the context of this Review.
Aromatic polyester dendrimers were the first polyester dendrimers to be described in 1992, by Hawker and Fréchet.(17) With this work, they introduced the convergent synthetic route for the synthesis of dendrimers. Despite containing ester linkages, they are aromatic esters, with high hydrolytic stability. Thus, these structures can barely be regarded as biodegradable dendrimers. In 1996 the first study mentioning biodegradable dendrimers was reported, where the enzymatic degradation of aliphatic chiral polyester dendrimers based on (R)-3-hydroxybutanoic acid and trimesic acid was described ( Figure 1).(18) Since then, several other polyester dendritic structures have been proposed: 2,2bis(hydroxymethyl)propanoic acid (bis-HMPA)-derivatives (section 3.1), other aliphatic esters derivatives (section 3.2), and alternating polyesters (section 3.3). In this work, all the aliphatic polyester dendrimers will be considered biodegradable, even in those cases in which authors did not study their degradation profile.
In the following subsections, the design and synthesis of several biodegradable dendritic structures are presented.
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Monomers Based on bis-HMPA.
Since its first use, 2,2-bis(hydroxymethyl)propanoic acid (bis-HMPA) (Figure 2a) has been the main monomer of choice in the synthesis of biodegradable dentritic structures. Bis-HMPA is commercially available at low cost, and the resulting polyester dendrimers are non-immunogenic, nontoxic, and biodegradable:(19) all very attractive features when envisaging biological applications.
The first report on the synthesis of polyester dendrimers based on bis-HMPA was by Ihre, Hult, and Söderlind ( Figure 2c).(20) Since then, a considerable number of research groups proposed several alternative synthetic routes and different precursors using different protecting groups (Figure 2b), with the aim of improving and simplifying the synthesis of this family of biodegradable dendrimers until higher generations with good yields. Additionally, different functionalizations of the core and/or on the surface were reported by several authors to use these dendrimers for different purposes. (21) In 2002, Gillies and Fréchet described dendrimers consisting of two bis-HMPA dendrons covalently attached as a "bow-tie" ("bow-tie dendrimers", Figure 2d) aiming at water-soluble drug carriers. (22) In 2004, Vestberg et al. reported the synthesis of bis-HMPA dendrons up to G5 with porphyrins as core.(23) Porphyrins were used because of their potential applications in many areas, such as photodynamic therapy, nanosensors, and so forth.(24) Bis-HMPA dendrimers containing carboranes, which interest resides in their potential use in boron neutron capture therapy in cancer treatment and will be revisited in more detail in section 4, were synthesized by Parrott(21m) and Galie.(25) Gillies and Fréchet reported the synthesis of polyester dendritic bow-ties with a bis(adamantylurea)-glycinylurea system at the focal point of the bow-tie.(26) They prepared self-assembled polyester bow-tie dendrimers with various peripheral groups.
Bis-HMPA polyester dendrimers presenting cyclic carbonates on their periphery were also reported. The attraction of this approach is that carbonate groups react with amines, even in water, yielding bifunctional products easily. (27) Some studies that combined identical dendrons via the Diels-Alder reaction to furnish symmetrical dendrimers were reported.(28) In 2008, Kose et al. reported the first example of the synthesis of segment block dendrimers using the Diels-Alder-based synthetic strategy in order to get unsymmetrical dendrimers.(29) The cycloaddition is very attractive in dendrimer synthesis, since no reagents are required. Therefore, the resulting dendrimers are free of impurities such as toxic metals, which represent a problem for biological applications.
Several authors reported linear-dendritic block copolymers for different purposes including drug delivery, where the characteristics of biodegradable bis-HMPA dendrons are combined with linear polymers, also biodegradable in some cases. Polyester dendronized polymers (linear polymers having polyester dendrons at each repeating unit) that are expected to present an extended conformation were also reported by Grayson and Fréchet (based on a nondegradable linear polymer: poly(phydroxystyrene))(30) and Lee et al. (based on a degradable linear polymer: poly(ε-caprolactone) (PCL)).(31) Due to their high molecular weight and multivalency, these systems are also considered as promising vectors for drug delivery applications. The degradation half-life at 37 °C for the dendronized hydrophilic PCL was 2.5 days at pH 9.0 and 16 days at pH 7.4 (physiological conditions), while no changes in polymer molecular weight was observed under acidic conditions (pH 5.0, endolysosomal conditions).(31) These authors determined that, in fact, the dominant mode of degradation of these Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 ester bonds was under mildly basic conditions (pH 9.0). Additionally, the dendronized hydrophilic polymer was found to degrade much faster than the parent hydrophobic polymer. (32) Connal et al. explored the known "arm first" synthetic strategy to synthesize cross-linked star polymers, with bis-HMPA dendrons at the end of the arms. Bis-HMPA dendrons up to the G5 were synthesized and functionalized at the focal point with a single alkyl halide, capable of initiating polymerization of styrene by atom transfer radical polymerization (ATRP) (Figure 2f).(33).
In 2009, a series of well-defined and amphiphilic dumbbell-shaped triblock copolymers consisting of comb-shaped poly(l-lactide) (PLLA) end arms and linear PEG center block connected using bis-HMPA dendrons, with narrow molecular weight distributions and varied PLLA arm lengths, were presented ( Figure 2e).(34) These dendronized linear polymers provide an alternative dendritic architecture with a variety of aspect ratios accessible depending on the length of the PLLA arms and PEG block, and the generation of the dendrons. Efficient regulation of material properties and cell responses was achieved using this series of compounds, that suggests their potential for various regenerative medicine and tissue-engineering applications.(34) Subsequently, a series of well-defined dumbbell-shaped triblock copolymers consisting of linear PEG linked to the focal point of bis-HMPA dendrons and comb-like poly(ε-caprolactone) with different arm lengths were further used to prepare microspheres with potential application as carriers for controlled delivery of water-soluble drugs. (35) Recently, diblock and triblock dendron-polymer conjugates containing biodegradable bis-HMPA polyester dendron blocks and PEG were synthesized using the Diels-Alder "click" cycloaddition reaction by Gok et al. (36) Kempe et al. reported the synthesis, in a one-pot cascade reaction approach, of a bis-HMPA dendron functionalized at the focal point with poly(2-ethyl-2-oxazoline) (Pox). Pox is a polymer with similar properties to PEG, such as protein repellency and stealth behavior; thus, it is being extensively discussed as a potential alternative to the use of PEG. (37) Recently, Feliu et al. carried out a comprehensive assessment of the in vitro biocompatibility and degradability of a library of hydroxyl terminated bis-HMPA dendrimers of five different generations and their corresponding dendrons, that revealed excellent biocompatibility for these materials in the model systems used.(38) Besides the expected pH dependence of the degradation rate, in line with the previously reported data for bis-HMPA dendronized PCL,(31) it was found that the hydrolysis occurs first at the dendrimer periphery and progresses toward the core, through a mechanism of depolymerization. Additionally, sterically crowed G4-OH dendrimers were found to be extensively degraded after 2 days of incubation at pH 7.5. (38) Thus, despite the widespread use of these bis-HMPA-based dendritic structures, some issues regarding premature or undesired degradation still remain to be addressed. Namely, the unwanted degradation of the PEGylated polyester backbone observed during the attachment of certain drugs.(39) Also, a significant degradation after only 10 h under physiological conditions (pH 7.4 and 37 °C) was observed by van der Poll et al. for a G1 bis-HMPA-core dendrimer, which limits their potential use in certain biological applications. (16a) In order to solve this problem, these authors reported a synthesis that combines the biocompatibility and biodegradability of polyester dendrimers with the robustness of polyamide dendrimers, to yield a hybrid scaffold capable of translation into in vivo studies. In this manner, the degradation rate was significantly reduced, with the degradation profile for these esteramide hybrid dendrimers being prolonged up to 20 days. (16a).
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Other Aliphatic Ester Monomers
Other aliphatic ester monomers have been used to synthesize polyester dendrimers, although to a smaller extent than bis-HMPA. Carnahan and Grinstaff have reported polyester dendrimers coined as "biodendrimers", which are composed of natural metabolites and biocompatible with the human body. These authors developed several dendrimers based on only succinic acid, only adipic acid, and mixtures of the two (Figure 3a). (40) The properties of the mixed dendrimers depends on the composition of the outer generation layer.(40c) The esterification of the dendrimers with succinic acid monomethylallyl ether, and subsequent photochemical polymerization of the alkenes, yielded soft gels.(40a) These materials have been explored as corneal adhesives(41) and for cartilage repair.(42).
The synthesis of another family of biodegradable cationic dendrimers was proposed in 2006 ( Figure  3b). (43) The ester-amine dendrons and dendrimers synthesized in this work by the convergent method up to G3 and G2, respectively, were based on bis(2-hydroxy-ethyl)-amino]-acetic acid tert-butyl ester as the growing unit ( Figure 3b), with internal tertiary amines and protected terminal amines. The synthesized dendrons and dendrimers, with their terminal amines deprotected, were degradable in D2O at 37 °C. The degradation rates were found to be dependent on the generation number and the ability of the primary amines to access and to catalyze the ester group degradation. All dendrons were completely degraded within 30 days, while only 60% of the sterically crowded dendrimers were degraded over the same time period. (43) Twibanire et al. synthesized tribranched dendrons in order to prepare polyester dendrimers with denser layers than those derived from bis-HMPA and, therefore, more resistant under physiological conditions.(21g) The synthesis of polyester dendrimers using as starting materials benzyl acetoacetate and tert-butyl acrylate ( Figure 3c) were presented by Hirayama et al.(44) Similar dendrimers, also designed to be applied as drug delivery vectors, were prepared from 3-hydroxyacetophenone and its tert-butyldimethylsilyl ether. (45) Polyester dendrimers containing tertiary amines were prepared by Bouillon et al. (46) The amine groups can serve as buffers to neutralize the protons delivered from the ester hydrolysis; therefore, these poly(ester amine) dendrimers are very attractive as drug delivery vectors ( Figure 3d).
Another interesting contribution was the synthesis of polyester dendrimers presenting functional groups capable of orthogonal reactions,(47) rendering bifunctional dendrimers.(48) An AB2C dendron (Figure 3e.1) was used to originate dendrimers bearing alkyne units and hydroxyl groups (Figure 3e.2). A bifunctional dendrimer hosting azide and alcohol functionalities was also synthesized. (48) In 2013, Akiyama et al. presented divergent and convergent procedures for the synthesis of another type of poly(ester amine) dendrimers, having an adamantane structure as core, which allows that even low generation dendrimers have a globular structure.(49) Recently, Pahovnik and et al. also reported on the synthesis of different generation, water-soluble poly(ester amide) dendrimers with hydroxyl functional groups from an AB2 adduct of bis-HMPA and glycine as repeating unit and 1,1,1tris(hydroxymethyl)propane as core. These poly(ester amide) dendrimers were used for solubilizing an anti-diabetic drug.(50)

Alternating Polyester Dendrimers
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Ma et al. developed a facile synthesis of biodegradable alternating polyester dendrimers from sequential click coupling of asymmetrical monomers (2-[(methacryloyl)oxy]ethyl acrylate (MAEA) and cysteamine), with pendant methacrylate or amine groups that can be easily used for conjugations of drugs.(54) Later, Ye et al. simplified the synthesis by using a β-cyclodextrin core, from which polyester dendrimers with high molecular weights could be easily synthesized in fewer steps without intensive purifications. (14) Moreover, they carried out the in vivo evaluation of these biodegradable dendrimers as MRI contrast agents, the data of which will be further discussed in section 6. (14) 56) Dendrimers based on bis-HMPA were obtained through the photochemically induced addition of these macrothiols to alkene-terminated core molecules. Deprotection of the latent hydroxyls originated a hydroxyl-terminated dendrimer (Figure 4b.2) that can then be functionalizated further to give products with desired properties. (56) An efficient convergent synthetic route alternating esterification with light-promoted addition of a thiol to an alkyne was described by Chen et al. This dendrimer was shown to effectively bind the anticancer drug cis-dichlorodiammineplatinum(II).(57) Rosen et al. presented another alternating synthetic polyester dendrimers route for synthesizing dendrimers containing α-bromo esters, which can polymerize by a single electron transfer living radical polymerization and thus produce star polymers with low generations of dendrimer units at the center and on the periphery.(58) Downing et al. reported that acyloxysilyl bonds (silyl esters) can be incorporated into dendritic structures as an easily degradable bond. (59) In 2013, Khoee et al. published the preparation of pH-susceptible linear-dendritic-linear block copolymers from poly(ε-caprolactone), asymmetric poly(ester amine) dendrons (G1-G3), and PEG.(60) Their hydrolytic degradation was investigated at two pHs (5.8 and 7.4) at 37 °C. They observed that the copolymers with the higher-generation dendrons degraded more slowly than those with lower generations. Furthermore, in this case, the degradation rate at pH 5.8 is faster than at pH 7.4 for all generations. These amphiphilic systems can self-assemble and form micelles in water. Thus, they designed nanoparticles containing magnetite coated with these block copolymers for encapsulating hydrophobic drugs, such as quercetin. Their sensitivity at lower pHs is an attractive feature for using these nanoparticles for drug delivery in vivo.(60)

Degradable Dendrimers Not Based on Ester Bonds
An approach to obtain polyacetal systems using sequential transacetalation and protectiondeprotection techniques has been proposed by Fuchs and Lemcoff for the preparation of macromolecular polyacetals that were subsequently applied to obtain new dendrimers, which may undergo hydrolysis to effectively "unzip" the dendrimer to polyfunctional macromolecules or degrade it altogether. (61) Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/

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Using an iterative and divergent approach, Zimmerman and co-workers synthesized dendrimers from degradable 1,3,5-triazaadamantanes (TAA) monomers at each branch point (Figure 4c).(62) Contrary to some polyester dendrimers, TAAs are stable under basic and physiological conditions (pH 7.4), but hydrolyze rapidly (half-life lower than 30 min) under acidic conditions (e.g., endosomal pH) to give basic and well-defined byproducts. (62a) In the following sections, the applications of biodegradable dendritic structures as vectors of both drugs and nucleic acids, as well as their function as macromolecular contrast agents in MRI, are revisited.

Biodegradable Dendritic Structures in Drug Delivery
One of the first applications of dendrimers in the biomedical field was as drug delivery vectors, since they can easily transport drug molecules in their interior and/or tethered to their terminal surface groups.(5a, 63) The advantages of using dendrimers as drug carriers are multiple and include the following: (a) the possibility of being designed to carry hydrophobic or hydrophilic drugs, protecting them from degradative processes or unwanted interactions with biological molecules; (b) the capacity to simultaneously release two or more drugs at the site of interest allowing the tuning of pharmacodynamics; (c) the potential to modify the dendrimer surface to confer hydrophilicity to the dendrimers, to help them to surpass important biological barriers (such as those imposed by the mononuclear phagocyte system or cellular membranes), or to endow them with chemical ligands that are recognized by cell receptors and actively target specific types of cells or tissues; (d) the chance to play with the dendrimer size (generation) to modulate the dendrimer's blood circulating time and/or passively target solid tumors through the enhanced permeability and retention (EPR) effect; (e) the ability to achieve a controlled delivery of the drugs by controlling the dendrimer/drug interactions or by developing stimuli-responsive systems; and (f) the possibility to use dendrimers as theranostic platforms through their combination with chemical entities/nanoparticles suitable as contrast agents in bioimaging (this point will be further explored in section 6).(5a, 63) Biodegradable dendrimers show all the mentioned potentialities of dendrimers for drug delivery, further presenting the advantage of being transformed under physiological conditions into small-size products that may be metabolized or excreted from the body. In this context, the dendrimers and their products of degradation should show a low level of toxicity, as is the case for polyester dendrimers.(19, 38, 64) About 12 years ago, in a representative work, Padilla De Jesús evaluated the toxicity both in vitro and in vivo of several dendritic structures based on bis-HMPA.(64) All the structures were shown to have a low cytotoxicity and a high tolerability upon administration in mice by intravenous (i.v.) bolus injection. One of these structures, a high-molecular-weight 3-arm PEG-dendrimer hybrid (that exhibited the longest circulatory half-life) was conjugated to doxorubicin by means of an acid-labile hydrazone linkage. In vitro experiments showed that the system was able to release the drug as a function of pH with a higher release at pH below 6. Results also indicated that the cytotoxicity of the drug was significantly reduced after linking it to the dendrimer, demonstrating that the use of a carrier alters the pharmacokinetics and the distribution of the drug. The design of the dendrimer structure to prevent rapid and premature degradation was also revealed as an important feature toward obtaining an effective drug delivery system. Later, the same group reported a study where orthogonal "bow-tie" polyester dendrimers were prepared with PEG arms connected to one of the polyester dendrons via degradable carbamate bonds (Figure 2d). (19) Here, the polyester dendron was conjugated with the Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 drug and, while complete ester hydrolysis alone should release the bis-HMPA monomers, carbamate hydrolysis should release the individual PEG arms. A library of eight polyester dendrimer-PEG bow-tie hybrids was prepared (molecular weights from 20 000 to 160 000, and number of PEG arms ranging from two to eight) and evaluated as controlled drug release systems. In this work, as expected, the high-molecular-weight carriers (>40 000) exhibited longer circulation half-lifes. Furthermore, the in vitro degradability of the G3 bow-tie with 10 kDa PEG was followed for 15 days at pH 9.0, pH 7.4, and pH 5.0 and 37 °C. The reported results were in line with the ones reported by Lee et al. for the bis-HMPA dendronized PCL.(31) Additionally, the obtained results showed that at pH 5.0 only the carbamate bonds degrade, while at pH 7.4 the ester bond present in the bis-HMPA dendron backbone also degrades. (19) A subsequent study showed the effectiveness of these carriers as drug vehicles.(65) Indeed, a single injection of a high-molecular-weight dendrimer-PEG-doxorubicin conjugate substantially inhibited the progression of a doxorubicin-insensitive C-26 colon carcinoma upon i.v. administration to BALB/c mice. A PEGylated dendrimer based on a polyester-polyamide hybrid backbone was also described for doxorubicin transport through conjugation. (16a) As previously mentioned at the end of section 3.1, the design of the carrier was chosen to diminish destructive side reactions during dendrimer synthesis and premature degradation, while a suitable biodegradability is equally maintained. The anticancer efficiency of the dendrimer-doxorubicin conjugate was compared to that of Doxil using equal dosages in the treatment of C26 murine colon carcinoma. In this work, statistically equivalent results were obtained with the two systems with most mice tumor-free at the end of the two-month experiment, with the test group animals indicating that the ester-amide dendrimer may exhibit less toxicity than Doxil at equivalent doxorubicin dosages. (16a) The encapsulation of drugs inside dendritic structures is a common strategy in drug delivery that can help in the solubilization of hydrophobic drugs such as the camptothecins that have low water solubility but high anticancer efficiency. Morgan et al. used one of their "biodendrimers", the one based on glycerol and succinic acid, for the encapsulation of 10-hydroxycamptothecin and 7-butyl-10aminocamptothecin.(66) The cytotoxicity of the dendrimer-drug complexes toward four different human cancer cell lines was analyzed showing low IC50 values. Importantly, cellular uptake and drug retention were increased in MCF-7 cells when using the prepared dendrimer.(66) In another study, Dhanikula and Hildgen investigated the influence of the architecture of polyester-co-polyether (PEPE) dendrimers on the encapsulation and release of the anticancer drug methotrexate. (67) In vitro experiments showed that the different dendrimers were biocompatible and presented a good capacity to encapsulate this drug. An increase in the number of branches and in the size of internal cavities was shown to enhance the encapsulation capacity, while the absence of aromatic rings as branching units substantially decreased the loading capacity of PEPE dendrimers. The authors concluded that the mechanisms of encapsulation involved physical entrapment, weak hydrogen bonding, and hydrophobic interactions. (67) Interestingly, thermoreversible hydrogels were prepared by Namazi and Adeli from dendronized polymers based on citric acid (dendron part) and PEG (linear part).(68) It should be highlighted that several hydrophobic anti-inflammatory drugs have been successfully encapsulated inside the formed gels. Lundberg et al. presented bis-HMPA dendrons of different generations (G0-G4) as functionalized macroinitiators for the construction of sophisticated amphiphilic dendritic-linear hybrid materials with hydrophobic poly(ε-caprolactone) at the chain-ends and a single strain of hydrophilic PEG at the core.(69) These were utilized for the fabrication of drug loaded micelles as well as the development of isoporous membranes.
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Though still at a preliminary stage, "disassembled", "cleavable", or "self-immolative" dendrimers (SIDs) that respond to an external stimulus (e.g., pH variation, enzymatic action, catalytic antibodies, among others) constitute very promising systems for drug delivery.(70) These dendrimers have a unique structural degradable backbone, the most common cleavage functionalities of which are esters and carbamates, that allows a cascade decomposition upon a simple triggering event. If drug molecules are incorporated as the tail units in these dendrimers and a suitable stimulus is used as the trigger, a multi-prodrug is generated that may be activated by a single cleavage event. The advantages associated with these dendritic prodrugs are significant when compared with monomeric prodrugs in inhibition of tumor cell growth. (71) Biodegradable dendrimers have also been used in the delivery of boron-10 for neutron capture therapy (NCT) of cancer. For this, it is crucial to ensure an adequate boron concentration at the target sites, and as such, high boron content species (like carboranes) can be used in the design of the carriers. The polyester dendrimers based on bis-HMPA proposed by Parrott et al. incorporated several carboranes within its structure (section 3.1).(21m) Fourth-and fifth-generation dendrimers that contained 4, 8, and 16 carborane cages were prepared. The irradiation of these dendrimers with thermal neutrons resulted in emission of γ radiation, thus showing the occurrence of boron neutron capture events and the potentiality of using these dendrimers in cancer treatment.(21m)

Biodegradable Dendritic Structures for Nucleic Acid Delivery
Over the past decades, gene therapy has emerged as a promising therapeutic approach to prevent and treat several diseases/conditions. Its underlying principles are the modulation of gene and/or protein expression of the host cells following the introduction of exogenous nucleic acids (DNA, RNA, or singlestranded oligonucleotides) into somatic cells. (72) Nucleic acids (NAs) are susceptible to degradation by serum endonucleases, further decreasing their already low internalization when administered naked.(1e) Moreover, following cellular uptake, NAs are susceptible to additional degradation both in the lysosomal/endosomal pathway and in the cytoplasm.(72, 73) Thus, most of the gene therapy strategies proposed so far rely on delivery vectors that, ideally, should contribute to the overcoming of different extra-and intracellular barriers in order to efficiently deliver NAs into cells with minimal toxicity.(72, 73) Briefly, the main hurdles toward a successful gene therapeutic intervention include the following: (a) NA degradation by endonucleases present in the extracellular milieu, (b) cellular internalization, (c) endosomal escape, (d) NA release from the vector and access to the cytoplasmic or nuclear target, and (e) vector intra-and extracellular accumulation. Nuclear delivery, necessary in the case of DNA(72, 73) or certain oligonucleotides,(74) further requires that the NA crosses the nuclear membrane. Additionally, the delivery vector must avoid unspecific binding to serum proteins, preventing aggregation.(5c, 72a, 73) NA vectors are divided into two main categories: viral and nonviral vectors. (72a, 73) Despite the high transfection rates observed for viral vectors, insertional mutagenesis and immunogenicity together with both low scale production and carrying capacity are delaying their application as safe and viable gene therapy vectors. (72a, 73, 75) Consequently, these obstacles have drawn much attention to the development of nonviral vectors, such as lipids, polymers, and dendrimers.(1e, 72a, 73) A common feature among these has been their cationic nature. The same characteristics that make dendrimers attractive platforms for drug delivery are equally important when it comes to the delivery of therapeutic NAs. Their adaptable and tunable chemistry together with the high density of functional groups allows the design of an almost unlimited number of molecules and the conjugation of several ligands tailored to attain efficient Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 gene delivery.(5c, 63a) Nevertheless, much research is needed for these to progress into preclinical and clinical developments.
Cationic dendrimers are able to complex NAs through electrostatic interactions between their positively charged terminal groups and the negatively charged phosphate groups of the NA, originating dendriplexes.(5c, 72a) This process occurs in a concentration-dependent manner and is also affected by the medium properties, such as pH, temperature, and ionic concentration.(5c) Even though several groups reported the success of dendrimers as NA delivery vehicles,(7b, 7h, 76) the nonbiodegradability of the dendritic structures used remains a drawback yet to be solved.(6a, 8d, 63a) As previously mentioned, the ideal gene delivery vehicle should be biodegradable to prevent bioaccumulation and subsequent cytotoxicity.  (77a) The efficiency of DNA binding increased in a generation dependent manner due to the enhanced multivalency. Degradation studies showed that the G3 dendrimer was stable at pH 5.0, but degraded at the physiological pH 7.4 on the time scale of 8 days.(77a) In a slightly different approach, Barnard et al. replaced the benzyl ester protecting group in G2 dendrons by hydrophobic units (cholesterol and hydrocarbon chains) to promote their controlled self-assembly rendering a system with higher multivalency, which significantly enhances the DNA binding. Furthermore, they modified the surface groups with N,N-di(3aminopropyl)-N-(methyl)amine (DAPMA), a triamine that showed lower cytoxicity than spermine.(77b) As expected, these dendrons remained intact at pH 5.0 and degraded at pH 7.5. Even though cholesterol-functionalized dendrons were shown to have lower degradation rates than the hydrocarbon-functionalized ones, every dendron was completely degraded after a period of 6-10 h.(77b) The reported G2 dendrons could efficiently complex DNA and undergo cellular internalization, yet a low transfection efficiency was observed. The authors hypothesized that, at the lower endosomal pH, or when bound to DNA, the degradation of these dendrons becomes ineffective on the transfection time scale, which could explain their poor transfection performance.(77b) The same group further modified the cholesterol-functionalized G2 dendrons with small PEG chains (triethylene glycol and octaethylene glycol).(78) PEG addition was found to affect size and zeta potential properties of the resulting dendriplexes and enhanced DNA binding to different extents, with a higher binding affinity observed for the structures based on the longer PEG chain.
More recently, Barnard and et al. have reported a different strategy aimed at improving DNA release and dendron degradation.(79) They synthesized self-assembly disulfide-linked dendron nanoparticles, where the cholesterol groups were attached to the DAPMA-terminated G2 bis-HMPA dendrons by an S-S linkage. In vitro, upon addition of dithiothreitol, nanoparticles where shown to undergo disassembly in 1 h due to cleavage of the disulfide linkage, which consequently led to loss of selfassembled multivalent binding, triggering DNA release ( Figure 5). After that, the remaining fragments undergo further degradation over longer time scales (24 h), which validates a controllable doubledegradation process.(79) Disulfide linkers are widely used in the design of nonviral gene delivery vectors due to their enhanced degradability in the reductive environment of the cytoplasm. (72) Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/

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The success of gene therapy is highly dependent on the development of efficient and safe NA delivery vectors. Additionally, it is extremely important to develop new biodegradable and biocompatible vectors to prevent both bioaccumulation and cytotoxicity, and easy clinical translation. Such vectors could arise from newly developed dendritic agents, which allow an almost unlimited combination of structures and architectures together with the ability to multiconjugate several target-molecules.

Biodegradable Dendritic Structures as Magnetic Resonance Imaging Contrast Agents
Currently, medical imaging plays a major role in health care management as one of the most efficient techniques for early and accurate diagnosis and treatment follow-up, expectedly leading to more successful treatment. As a noninvasive technique, MRI allows high spatial resolution and both physiological and anatomical information without resorting to harmful ionizing radiation. However, both its low sensitivity and target-specificity remain significant limitations. (5d) MRI is based on the interaction of protons (mostly water protons) with each other and their surroundings. Upon the application of strong magnetic fields, the magnetic moments of protons will orient themselves in the direction of the magnetic field.(80) After disruption of this magnetic alignment they will return to the previous state in a process described by two relaxation rates: the longitudinal and the transversal relaxations, characterized by the time constants T1 and T2, respectively.(80, 81) The relaxation rates vary from tissue to tissue, which gives rise to the image contrast in MRI. However, the inherent differences in relaxation times among tissues are not always enough to generate an adequate contrast. Thus, it is rather common to use exogenous contrast agents (CA), which act by reducing T1 and T2 through interaction with the surrounding water protons.(5d, 80, 81) The ability to decrease T1 and T2 is measured by the r1 and r2 relaxivities, respectively (r1 = 1/T1; r2 = 1/T2). (80,81) Contrast agents can be classified by their biodistribution, magnetic properties (paramagnetic and superparamagnetic), and effect on the image contrast, which can be positive (T1 reduction) or negative (T2 reduction), increasing or decreasing the signal intensity, respectively.(82) Paramagnetic agents are predominantly T1-reducing CA, while superparamagnetic usually originate T2 reductions.(82) The most widely used CAs are small molecules based on paramagnetic gadolinium(III) chelates, such as Gd(III)-DTPA (DTPA = diethylenetriaminepentaacetic acid, Magnevist) and Gd(III)-DOTA (Dotarem).(5d, 83) Administered prior to the MR imaging, these CAs are known for their contrast enhancement. Yet, their low molecular relaxivity, nonspecificity, and fast renal clearance require the usage of potentially harmful high doses, which clearly limits image quality.(5d) Conjugation of Gd(III)chelates with polymers,(84) proteins,(85) dendrimers,(5d) or other scaffolds(86) can enhance relaxivity and blood circulation times improving image contrast.(1f) Due to their multivalency, dendrimers represent one of the most appealing platforms to carry multiple CAs moieties.(5d) Moreover, their branched structure brings an extra rigidity and the possibility to graft target molecules, offering a unique opportunity to enhance site-specific image contrast ( Figure 6). (81) The applicability of dendrimers as MRI contrast agents (dendritic CAs, DCAs) was first demonstrated by Wiener et al. where a PAMAM dendrimer conjugated with a Gd(III)-DPTA showed an ionic relaxivity of 34 mM-1 s-1, meaning a 6-fold higher relaxivity compared to that of Gd(III)-DTPA alone (5.4 mM-1 s-1).(87) Additionally, they observed that these novel structures were able to carry more Gd(III) ions than other existing macromolecules at the time, which could possibly be increased at higher generations. Although the macromolecular CAs developed so far have been yielding good image Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 contrast and enhanced sensitivity, the large sizes decrease renal excretion, which in turn significantly increases the possibility of bioaccumulation and further release of toxic Gd(III) ions. (5d, 83, 88) In fact, some studies have reported that nephrogenic fibrosis and other severe diseases were associated with the use of certain Gd(III) chelates, particularly in patients with impaired renal function.(89) So far, only Gadomer-17, a poly(lysine)-based dendritic CA bearing 24 Gd(III)-DOTA chelates, has advanced into clinical evaluation due to its almost exclusive intravascular biodistribution and rapid renal clearance. (90) Thus, recent efforts have been made to develop biocompatible and biodegradable DCAs (Table 1). These maintain the nondegradable DCAs characteristics, such as blood circulation times and enhanced image contrast, and at the same time can be easily degraded under physiological conditions into smaller byproducts preventing accumulation and subsequent toxicity.  14) G0 had a relaxivity of 10.6 mM-1 s-1, while G1, G2, and G3 showed similar relaxivity values around 11.7 mM-1 s-1. This phenomenon was attributed to the comparable internal flexibility of the higher generations, which hindered the expected rise of the relaxivity with the generation. Again, degradation studies, using the G3 dendrimer, showed that at mildly acidic pH little or no degradation was detected, while at pH 7.4 only 70% of the ester bonds remained intact 20 h post-incubation. This percentage decreased to 35% in the presence of an esterase. For in vivo studies the authors introduced zwitterions into the dendrimer to enhance solubility. The zwitterionic G2 DCA was shown to have higher blood circulation times and image improvement than the clinically used Magnevist. Additionally, this dendrimer was shown to promote lower long-term Gd(III) accumulation in several tissues when compared to the nondegradable dendritic MRI agent PAMAM-G6-DO3A-Gd(III) (DO3A = 1,4,4,7,. (14) More recently, molecular MRI has also drawn attention due to its potential use in the dissection of disease mechanisms and other events at the cellular and subcellular levels, which can be achieved by combining CAs with target molecules.(5d, 96) In 2012, the G2 polyester dendrimer reported above was further functionalized with PEG and folic acid (FA) to target tumor cells overexpressing folic acid receptors.(14, 89a) PEGylation resulted in enhanced relaxivity due to the increasing solubility and Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 complex stiffness. FA-PEG-G2-DTPA-Gd(III) had a higher and longer tumor image contrast than that of Magnevist and a much lower Gd(III) accumulation compared with nondegradable contrast agents, due to dendrimer degradation. Degradation studies showed that 30% of the ester bonds of this DCA hydrolyzed at pH 7.4 against 7% at pH 5.0 after 10 h. The hydrolyzed ester bonds at pH 7.4 were shown to increase to 47% in the presence of an esterase.(89a) Despite the great performance of this DCA, the need for solubilizing groups might hamper translation to the clinic.(93) In order to overcome this, the same team developed a new DCA based on the same polyester dendrimer, but with a tris(2aminoethyl)amine core to increase solubility without further modifications (PEGylation), hence preventing high molecular weights and size distribution.(93) As expected, they observed that r1 increased from 10.2 to 17.5 mM-1 s-1 when going from G1 to G5 dendrimer, demonstrating a generation-dependent relaxivity. Interestingly, in vivo MRI revealed major biodistribution differences between the G2 and G5 dendrimers, as the first enhanced tumor contrast whereas the second enhanced liver signal. The smaller size of the G2 allowed improved tumor imaging due to the passive tumor targeting via the EPR effect.(93, 97) As expected, G2-DTPA-Gd(III) displayed pH-dependent degradability, being stable at pH 5.0 and degraded at pH 7.4 (20 h post-incubation, 80% of the ester bonds were degraded). (93) Biodegradable dendritic-based MRI contrast agents have already proven to be a promising alternative to the currently available CAs. Even though the significant molecular weight increase could be a limitation to the clinical setting, it remains true that lower amounts of CA would be necessary due to the relaxivity increase. Therefore, further testing is needed to formulate an adequate molecular weight/relaxivity ratio. Success could rely on the optimization of already existing systems, as well as on the development of new biodegradable and biocompatible dendritic platforms. Moreover, the combination of several MRI labels and target-specific molecules in the same dendritic scaffold could give rise to better and more accurate diagnostics.

Conclusions and Future Perspectives
The use of degradable polymeric systems has a wide perspective in several established and emerging biomedical technologies, including controlled-release systems for targeted drug delivery, antibacterial drugs, and tissue regeneration. As the demand increases, a higher level of control over the structure, properties, and development of degradable materials is being pursued.
Dendrimers allow this coveted structural control. Among these, biodegradable dendrimers represent an attractive class of nanomaterials. They possess two major advantages over the conventional dendrimers, which confers an additional inherent "smartness" to these systems: (a) multiple covalently bound bioactive molecules can be site-specifically released from the targeted dendrimer by a single cleaving step, and (b) they are degraded and therefore can be easily eliminated from the body avoiding the toxicity related to the accumulation of synthetic materials in cells/tissues. Consequently, biodegradable dendrimers are expected to surpass the limitations of the most used nondegradable systems. Even so, the design and synthesis of biodegradable dendritic structures soluble in water with precise branching, molecular weight, monodispersion, and with a suitable pharmacodynamics continues to be a challenge. Other important key aspects are to overcome the undesired degradation of the degradable backbone observed during the attachment of certain functional groups and/or bioactives, as well as to limit the premature hydrolysis of the backbone and/or functional moieties in order to ensure their efficient application. In fact, these hurdles may explain the reduced number of reports where biodegradable dendrimers are applied for a particular function, in comparison with the higher number of works reporting only the design and synthesis of biodegradable dendrimers. This will Version: Postprint (identical content as published paper) This is a self-archived document from i3S -Instituto de Investigação e Inovação em Saúde in the University of Porto Open Repository For Open Access to more of our publications, please visit http://repositorio-aberto.up.pt/ A01/00 require considerable effort in the development of new synthetic routes for the development of these systems.
Nevertheless, once these drawbacks are surpassed, biodegradable dendrimers are expected to recast the existing therapeutic practices. The simultaneous combination of multivalency and biodegradability with precise architectures will definitely make dendrimers a greater platform, versatile for many biomedical applications as revised here.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Psychology

text:
Sensory Neurons Arouse C. elegans Locomotion via Both Glutamate and Neuropeptide Release

C. elegans undergoes periods of behavioral quiescence during larval molts (termed lethargus) and as adults. Little is known about the circuit mechanisms that establish these quiescent states. Lethargus and adult locomotion quiescence is dramatically reduced in mutants lacking the neuropeptide receptor NPR-1. Here, we show that the aroused locomotion of npr-1 mutants results from the exaggerated activity in multiple classes of sensory neurons, including nociceptive (ASH), touch sensitive (ALM and PLM), and stretch sensing (DVA) neurons. These sensory neurons accelerate locomotion via both neuropeptide and glutamate release. The relative contribution of these sensory neurons to arousal differs between larval molts and adults. Our results suggest that a broad network of sensory neurons dictates transitions between aroused and quiescent behavioral states.

Author Summary
Animals switch between periods of behavioral arousal and quiescence in response to environmental, developmental, and circadian cues. Little is known about the circuit mechanisms that produce these behavioral states. During larval molts, C. elegans exhibits a sleeplike state (termed lethargus) that is characterized by the absence of feeding and profound locomotion quiescence. We previously showed that mutants lacking the neuropeptide receptor NPR-1 exhibit increased arousal during larval molts, which is in part mediated by increased secretion of an arousal peptide (PDF-1). Here, we compare the circuits regulating arousal in larval molts and adults. We show that a broad network of sensory neurons arouses locomotion but that the impact of each neuron differs between lethargus and adults. We propose that this broad sensory network allows C. elegans to adapt its behavior across a broad range of developmental and physiological circumstances.

Introduction
Animals undergo periods of behavioral quiescence and arousal in response to changes in their environment and metabolic state. Arousal is defined as a state of heightened responsiveness to external stimuli coupled with increased motor activity whereas quiescence is associated with diminished responsiveness and motor activity [1]. Quiescence and arousal can persist for minutes to hours. Arousal is associated with fear, stress, hunger, and exposure to sexual partners [1], while quiescence is associated with sleep and satiety [2]. Relatively little is known about the specific circuit mechanisms leading to arousal or quiescence. In particular, it is unclear if similar mechanisms mediate quiescence and arousal in response to different cues, or at different times during development. To address this question, we have analyzed arousal and quiescence of C. elegans locomotion.
Mutants lacking NPR-1 Neuropeptide Y (NPY) receptors have been utilized as a model for generalized arousal. NPR-1 inhibits the activity of a central sensory circuit that is defined by gap junctions to the RMG interneuron [14]. In npr-1 mutants, responses mediated by the RMG circuit (e.g. pheromone and oxygen avoidance) are exaggerated, and this heightened acuity is associated with exaggerated locomotion (both during lethargus and in adults) [11,[14][15][16]. Mutations that increase (e.g. npr-1) and decrease (e.g. tax-4 CNG and osm-9 TRPV) RMG circuit activity are associated with locomotion arousal and quiescence respectively [11,14,17,18].
We previously showed that locomotion quiescence during lethargus is dramatically reduced in npr-1 mutants and that this effect requires increased RMG sensory activity [11]. Subsequent studies showed that in microfluidic chambers npr-1 mutants have modest defects in lethargus quiescence when sensory cues are minimized but that dramatic quiescence defects are observed following brief stimulation with light or vibration [19,20]. Taken together, these papers suggest that npr-1 mutants exhibit aroused locomotion as a consequence of enhanced sensory activity.
The arousing effects of the RMG circuit are mediated in part by secretion of a neuropeptide, pigment dispersing factor (PDF-1) [11]. Activation of PDF receptors (PDFR-1) in peripheral mechanosensory neurons enhances sensitivity to vibration, thereby accelerating locomotion. Thus, sensory evoked activity in the RMG circuit arouses locomotion during lethargus through changes in PDF-1 and PDFR-1 signaling. These results raise several interesting questions. Which specific sensory neurons are responsible for arousal? Does the RMG circuit regulate arousal via multiple outputs (i.e. in addition to PDF-1)? Does the RMG circuit function similarly during lethargus and in adults? Is diminished sensory acuity during lethargus required for behavioral quiescence?
Here we show that glutamatergic transmission promotes arousal, we identify glutamatergic neurons and glutamate receptors that mediate arousal, and we show that arousal occurs by distinct mechanisms in lethargus and adult animals.

Cholinergic transmission at NMJs is increased in npr-1 adults
Adult npr-1 mutants exhibit accelerated locomotion (Fig 1A-1C), as shown in prior studies [21]. Faster adult locomotion suggests that locomotion circuit activity has been altered. Consistent with this idea, npr-1 mutant adults have enhanced sensitivity to the paralytic effects of a cholinesterase inhibitor (aldicarb) (Fig 1D-1F and S2A Fig) [22], indicating increased excitatory transmission at neuromuscular junctions (NMJs). To more directly assess changes in synaptic transmission, we recorded miniature excitatory post-synaptic currents (mEPSCs) in body muscles, which are evoked by acetylcholine (ACh) release at NMJs. The mEPSC rate observed in npr-1 adults was significantly higher than in wild type controls while mEPSC amplitudes were unaltered (Fig 1G-1I). Faster mEPSC rates suggest that ACh release from motor neurons was increased whereas unaltered mEPSC amplitudes imply that muscle responsiveness to secreted ACh was unaffected. By contrast, neither ACh release evoked by depolarizing motor neurons with a stimulating electrode (evoked EPSCs), nor transmission at GABAergic NMJs (assessed by miniature inhibitory post-synaptic currents, mIPSCs) was altered in npr-1 mutants (S1 Fig). This constellation of electrophysiological defects suggests that tonic ACh release (assessed by mEPSC rate) was enhanced in npr-1 mutants, whereas other forms of neurotransmitter release (evoked ACh release and tonic GABA release) were unaffected. Enhanced tonic ACh release at NMJs could account for the accelerated locomotion rate observed in npr-1 adults.

Enhanced cholinergic transmission in npr-1 adults is caused by increased sensory activity
Prior studies showed that several behavioral phenotypes exhibited by npr-1 mutants are caused by enhanced sensitivity to environmental cues. In particular, sensory responses mediated by the RMG circuit are enhanced in npr-1 mutants [14,17,18] and this enhanced sensory acuity is required for accelerated locomotion rates during lethargus [11,20]. We did several experiments to determine if enhanced RMG circuit activity is also required for increased cholinergic transmission in npr-1 adults. A transgene restoring npr-1 expression in the RMG circuit (using the flp-21 promoter) rescued the accelerated locomotion (Fig 1B), enhanced aldicarb sensitivity ( Fig 1D and S2A Fig), and faster mEPSC rate (Fig 1G-1I) defects of npr-1 adults. By contrast, an npr-1 transgene expressed in GABAergic neurons lacked rescuing activity (Fig 1D-1H). These results indicate that NPR-1 acts in the RMG circuit to slow adult locomotion. Similarly, mutations inactivating ion channels required for sensory transduction (TAX-4/CNG and OCR-2/TRPV) in the RMG circuit suppressed the npr-1 adult locomotion (Fig 1C), aldicarb sensitivity ( Fig 1E and 1F and S2B and S2C Fig), and mEPSC rate (Fig 1J and 1K) defects. Collectively, these results suggest that the accelerated adult locomotion exhibited by npr-1 mutants is caused by heightened activity in the RMG sensory circuit and, consequently, corresponds to an aroused state.

Inactivating PDF signaling does not prevent aroused locomotion in npr-1 adults
We previously showed that the lethargus quiescence defects exhibited by npr-1 mutants are caused by increased secretion of Pigment dispersing factor (PDF-1) by cells in the RMG circuit [11]. Because PDF-1 secretion is also increased in npr-1 adults [11], we tested the idea that the hyperactive adult locomotion of npr-1 mutants is also caused by increased PDF signaling. Contrary to this idea, we found that pdf-1 and pdfr-1 (PDF Receptor-1) mutations reduced but did not eliminate the aldicarb hypersensitivity (Fig 2A and 2B and S2D and S2E Fig), the accelerated locomotion (Fig 2C), and increased mEPSC rate (Fig 2D and 2E) defects of npr-1 adults. Collectively, these results suggest that additional excitatory outputs from the RMG circuit (i.e. beyond PDF-1) must contribute to the aroused locomotion of npr-1 adults.
Glutamate released by sensory neurons is required for npr-1 locomotion and EPSC defects Many C. elegans sensory neurons are glutamatergic, including two neurons in the RMG circuit (ASH and ASK) and the body touch neurons [23]. To determine if glutamate release by sensory of trials is indicated for each genotype. Full time courses of aldicarb-induced paralysis are shown in S2A-S2C Fig. (D) The npr-1 aldicarb hypersensitivity was rescued by transgenes expressing NPR-1 in the RMG circuit (RMG rescue, flp-21 promoter) but not by those expressed in GABAergic neurons (GABA rescue, unc-25 and unc-30 promoters). (E-F) The npr-1 aldicarb hypersensitivity was blocked by mutations inactivating TAX-4/CNG channels or OCR-2/ TRPV channels. (G-L) mEPSCs were recorded from body wall muscles of adult worms for the indicated genotypes. Representative traces of mEPSCs (G and J) and summary data are shown (H, I, K, and L). (G-I) The npr-1 cholinergic transmission defect was rescued by transgenes expressing NPR-1 in the RMG circuit (RMG rescue, flp-21 promoter) but not by those expressed in GABAergic neurons (GABA rescue, unc-30 promoter). (J-L) The npr-1 cholinergic transmission defect was abolished by mutations inactivating TAX-4 or OCR-2. The number of animals analyzed is indicated for each genotype. Error bars indicate SEM. Values that differ significantly are indicated (*, p <0.05; **, p <0.01; ***, p <0.001; ns, not significant).  neurons is required for accelerated locomotion in npr-1 mutants, we analyzed mutations that inactivate the vesicular glutamate transporter (eat-4 VGLUT), which is primarily expressed in sensory neurons [23]. eat-4 VGLUT mutations blocked the increased motile fraction and locomotion speed of npr-1 mutants both during the L4-Adult (L4/A) molt (Fig 3A-3C) and in adults (Fig 3D and 3E). eat-4 mutations also blocked the hypersensitivity to aldicarb ( (Fig 3G and 3H) defects of npr-1 adults. Transgenes restoring EAT-4 expression in touch neurons and ASH neurons partially reinstated both lethargus (Fig 3B and 3C) and adult locomotion (Fig 3D and 3E) defects in eat-4; npr-1 double mutants, whereas transgenes expressed in ASK lacked rescuing activity (Fig 3B and 3C). eat-4 transgenes had no effect on lethargus quiescence in wild type animals (S3 Fig). These results suggest that glutamate released by ASH and touch neurons arouses locomotion in L4/A and adult npr-1 mutants.

ASH activity is associated with locomotion arousal
The preceding results suggest that ASH synaptic output arouses locomotion in npr-1 mutants. We did several additional experiments to test this idea. If altered ASH output were required for aroused locomotion, we would expect that npr-1 mutants lacking ASH neurons would have increased locomotion quiescence. To test this idea, we induced ASH cell death with a transgene that expresses the pro-apoptotic caspase CED-3. Killing ASH significantly decreased the L4/A motile fraction and locomotion rate in npr-1 mutants (Fig 4A-4C). By contrast, ASH ablation had little effect on the locomotion rate of npr-1 adults ( Fig 4D).
To determine if ASH activity is increased in npr-1 mutants during lethargus, we examined sensory-evoked calcium responses in ASH, using the genetically encoded calcium indicator Cameleon. ASH mediates avoidance responses to copper and hyper-osmotic stimuli. Consistent with a recent study [12], the magnitude of copper (Fig 4E and 4F) and glycerol-evoked (S4A and S4B Fig) calcium transients in ASH was significantly decreased during lethargus in wild-type animals. Decreased ASH responsiveness to copper and glycerol during L4/A lethargus was blocked in npr-1 mutants, whereas ASH responsiveness in adults was unaltered in npr-1 mutants (Fig 4E and 4F and S4A and S4B Fig). Transgenes expressing NPR-1 in the RMG circuit (using the flp-21 promoter) or in ASH (using the sra-6 promoter) reinstated the L4/A decrease in copper and glycerol-evoked ASH calcium transients in npr-1 mutants (Fig 4G and  4H and S4C and S4D Fig). These results suggest that NPR-1 acts in ASH to inhibit sensory responses and that increased ASH activity is required for accelerated locomotion of npr-1 mutants during lethargus but not in adults.
To determine if increased ASH activity is sufficient to arouse locomotion, we analyzed locomotion after artificially depolarizing ASH neurons. For this experiment, we utilized transgenic animals that express rat TRPV1 capsaicin receptors in ASH neurons [24]. In these animals, capsaicin treatment evokes ASH-mediated avoidance behaviors [24]. A 5-hour capsaicin treatment had little effect on L4/A motile fraction and locomotion velocity [11], whereas capsaicin treatment significantly accelerated adult locomotion and increased aldicarb sensitivity (Figs 4I-5J and S2G Fig). These effects were not observed in animals lacking TRPV1 expression in ASH neurons (Fig 4I and 4J). Thus, forced ASH depolarization was sufficient to arouse adult but not lethargus locomotion. Collectively, these results suggest that diminished and heightened ASH activity is associated with locomotion quiescence and arousal respectively; however, the magnitude of ASH's arousing effects differ between lethargus and adult animals.
GLR-2 AMPA receptors are required for the npr-1 lethargus defect Which glutamate receptors arouse locomotion in npr-1 mutants? Glutamate-activated cation channels, AMPA (GLR-1 and -2) and NMDA (NMR-1 and -2) receptors, mediate excitatory transmission at ASH-interneuron [25][26][27]. The npr-1 L4/A quiescence defect was abolished in glr-2; npr-1 double mutants (Fig 5A-5C), while glr-1 mutations had no effect (Fig 5D and 5E). By contrast, glr-1, glr-2, and nmr-1 mutations had little effect on npr-1 adult locomotion ( Fig  5F and S5 Fig). Similarly, glr-2 mutations did not block the increased mEPSC rate in npr-1 ASH activity is associated with locomotion arousal. Locomotion behavior during the L4/A lethargus (A-C) and in adults (D) of single worms whose ASH neurons were ablated by transgenic overexpression of CED-3 in ASH neurons (sra-6 promoter) was analyzed in the indicated genotypes. Animals were analyzed by fluorescence microscopy after locomotion recordings to determine if ASH neurons were ablated (1-2 ASH: animals with 1 or 2 ASH intact neurons; 0 ASH: animals lacking viable ASH neurons). Instantaneous locomotion velocity (A), average motile fraction (B), and average locomotion velocity (C-D) are plotted. The npr-1 locomotion defect during the L4/A lethargus, but not in adults, was partially suppressed in the transgenic animals in which both of ASH neurons were ablated (0 ASH). (E-H) Copper-evoked calcium transients in ASH were analyzed in L4, L4/A, and adults of the indicated genotypes using cameleon as a calcium indicator. Averaged responses (E, G), and the amplitudes of individual trials (F, H) are shown for each genotype. Each trace represents the average percentage change in YFP/CFP fluorescence ratio. The light tan rectangle indicates the duration for which 10 mM copper was adults ( Fig 5G). These results suggest that GLR-2 AMPA receptors are specifically required for the aroused locomotion during the L4/A lethargus in npr-1 mutants.
GLR-2 AMPA receptors act in AIA and DVA to mediate arousal Which synaptic targets of ASH and touch neurons mediate locomotion arousal? To address this question, we identified the neurons in which GLR-2 function is required. Aroused L4/A locomotion requires GLR-2 but not GLR-1 receptors; consequently, we reasoned that the relevant neurons are likely to express GLR-2 but not GLR-1. GLR-1 and GLR-2 are co-expressed in many neurons; however, a few GLR-2-expressing neurons lack GLR-1, including DVA (a stretch-activated neuron) and AIA (an interneuron in the head ganglia) [25][26][27]. The L4/A quiescence defect was partially restored in glr-2; npr-1 double mutants by transgenes expressing GLR-2 in DVA and AIA neurons, whereas transgenes expressed in the ventral cord interneurons (using the glr-1 promoter) failed to rescue (Fig 5B and 5C). Transgenic expression of GLR-2 in DVA or AIA had no effect on lethargus quiescence in wild type worms (S3 Sig). These results suggest that GLR-2 AMPA receptors expressed in AIA and DVA neurons arouse L4/A locomotion in npr-1 mutants. DVA receives direct synaptic input from the touch neuron PLM while AIA receives direct input from ASH [28]. Thus, increased transmission at ASH-AIA and PLM-DVA synapses could account for GLR-2's effects on locomotion rate. Because we only observed partial rescue by glr-2 transgenes expressed in AIA and DVA, it is likely the GLR-2 function is required in additional (as yet unidentified) neurons.
How do AIA and DVA arouse locomotion? AIA neurons provide synaptic input to ASK and ASI, both of which express PDF-1 [11,29]. Thus, heightened AIA activity could arouse locomotion by enhancing PDF-1 secretion. To assess the level of PDF-1 secretion, we analyzed PDF-1::YFP fluorescence in the endolysosomal compartment of coelomocytes, which are specialized scavenger cells that internalize proteins secreted into the body cavity [30,31]. Inactivating GLR-2 did not alter PDF-1::YFP fluorescence in coelomocytes in both adult and L4/A animals (Fig 6). These results suggest that the arousing effects of GLR-2 are not mediated by changes in PDF secretion. DVA neurons receive direct synaptic input from the PLM touch neurons [32], and secrete NLP-12 (a neuropeptide that accelerates locomotion) [33]. Thus, increased DVA activity could contribute to locomotion arousal in npr-1 mutants. Three results support this idea. First, PLM neurons exhibit enhanced touch-evoked calcium responses in adult npr-1 mutants (S6 Fig). Thus, PLM neurons have increased sensory acuity in npr-1 mutants, similar to the effect we previously showed for ALM neurons [11]. Second, inducing DVA cell death (with a CED-3 transgene) significantly reduced npr-1 locomotion rate during L4/A lethargus (Fig 5H-5J), but not in adults (Fig 5K). Third, DVA secretion of NLP-12 is significantly increased in npr-1 mutants [33], indicating increased DVA activity. These results suggest that PLM neurons provide enhanced excitatory input to DVA in npr-1 mutants, which promotes aroused L4/A locomotion.

Discussion
To investigate the circuit mechanisms for arousal, we analyzed the locomotion of npr-1 mutants in awake (adult) and quiescent (lethargus) states. Our results lead to five conclusions. First, multiple classes of sensory neurons contribute to arousal. Second, diminished sensory acuity is a  circuit mechanism for promoting behavioral quiescence. Third, glutamate and neuropeptides are utilized as excitatory outputs from sensory neurons to arouse locomotion. Fourth, different mechanisms are utilized to arouse locomotion at different times during development. And fifth, we provide further evidence that arousal mechanisms are conserved across phylogeny.

A broad network of sensory neurons contribute to arousal
Multiple classes of sensory neurons arouse locomotion during lethargus and in adults, including: mechanosensory neurons (ALM and PLM), a nociceptive neuron (ASH), a pheromone sensing neuron (ASK), and a stretch sensing neuron (DVA). Lethargus quiescence is accompanied by diminished sensory-evoked responses in ALM, PLM, and ASH (this study and [11][12][13]). PDF-1 secretion from ASK neurons is significantly reduced during lethargus, implying that ASK neurons also have diminished activity during lethargus [11]. npr-1 mutations prevent the dampened ALM (mechanosensory) and ASH (nociceptive) responses during lethargus and this was accompanied by decreased locomotion quiescence (this study and [11]). The arousing effects of npr-1 Fig 6. PDF-1 secretion is not altered in glr-2 mutants. PDF-1 secretion was analyzed in the indicated genotypes. YFP-tagged PDF-1 was expressed with the pdf-1 promoter. Representative images (A) and summary data (cumulative fraction) (B-C) are shown for coelomocyte fluorescence in L4/A lethargus and 1-day old adults of the indicated genotypes. PDF-1::YFP coelomocyte fluorescence was dramatically increased in npr-1 mutants during the L4/A lethargus and in adults as previously reported [11]. Mutations inactivating GLR-2 did not alter PDF-1::YFP coelomocyte fluorescence during L4/A lethargus (B) and in adults (C) in either wild type or npr-1 mutants. Scale bar indicates 10 μm. p values are indicated for each comparison (Kolmogorov-Smirnov test).
doi:10.1371/journal.pgen.1005359.g006 mutations are blocked (or diminished) by mutations that decrease sensory responsiveness (e.g. tax-4 CNG and osm-9 TRPV mutations) [11], or by ablating sensory neurons (e.g. ASH and DVA). Forced activation of ASH neurons arouses adult locomotion. Collectively, these results imply that a broad network of sensory neurons arouses locomotion, which allows C. elegans to adapt its behavior across a broad range of developmental and physiological circumstances.
Sensory gain control as a mechanism for producing quiescence and arousal NPR-1 promotes behavioral quiescence by diminishing the sensitivity of many sensory modalities. NPR-1 directly inhibits ASH responses and indirectly inhibits other sensory neurons (ALM, PLM, and DVA) via decreased glutamate and neuropeptide release. Thus, gating of sensory perception by NPR-1 provides a circuit mechanism for producing aroused and quiescent locomotion in C. elegans.
Our results do not exclude the possibility that additional mechanisms (beyond sensory gating by NPR-1) contribute to arousal and quiescence. Both quiescence (during lethargus) and arousal (following molts) persist in microfluidic chambers where many sensory cues are minimized [19]. In particular, oxygen tension is likely to be very low in these chambers, which would greatly diminish NPR-1's effects on behavior [15,16]. Thus, the quiescence and arousal exhibited in microfluidic chambers implies that additional mechanisms beyond NPR-1 must contribute to expressing these behavioral states. It will be interesting to determine if these NPR-1 independent mechanisms also act by gating sensory activity.

Sensory-evoked glutamate and neuropeptide release arouses locomotion
Sensory neurons release glutamate and/or neuropeptides in response to external cues, which then engage downstream motor circuits in behavioral outputs. Our prior study shows that sensory-evoked PDF-1 secretion promotes locomotion arousal by enhancing touch neuron responsiveness. Neuropeptides also mediate arousal in flies (PDF) [34], fish and mammals (orexin/hypocretin) [35,36].
Here we show that sensory evoked glutamate release also plays a role in arousal. Mutations inactivating the EAT-4/VGLUT decreased locomotion arousal in lethargus and in adults. EAT-4 is almost exclusively expressed in sensory neurons [23] and transgenes restoring EAT-4 in touch neurons and ASH neurons re-instates locomotion arousal in npr-1 mutants. These results suggest that sensory neurons utilize both glutamate and neuropeptides as excitatory outputs to arouse locomotion.
Our results suggest that exaggerated glutamate release at ASH-AIA and PLM-DVA synapses arouses locomotion during lethargus in npr-1 mutants. ASH and PLM neurons have enhanced sensory evoked activity in npr-1 mutants, which is expected to produce enhanced glutamate release at ASH-AIA and PLM-DVA synapses. GLR-2 receptors are expressed in AIA and DVA. glr-2 mutations block the aroused L4/A locomotion of npr-1 mutants and arousal is re-instated by transgenes expressing GLR-2 in AIA and DVA. Finally, calcium responses in AIA [14], and neuropeptide secretion from DVA [33] are both enhanced in npr-1 mutants, indicating that these neurons have increased activity. We observed only partial rescue of aroused locomotion by transgenes restoring EAT-4 expression in ASH and touch neurons or by those expressing GLR-2 in AIA or DVA; consequently, it is likely that glutamate released by other sensory neurons also contributes to the aroused L4/A locomotion in npr-1 mutants.
Much less is known about the role of glutamate in arousal in other systems. Glutamate release has widespread effects throughout the brain in mammals, which complicates the analysis of its effects on arousal. Microinjection of glutamate or AMPA into lateral hypothalamic area increased locomotor activity and duration of waking episodes in rodents [37,38], while microdialysis of CNQX, an AMPA receptor antagonist, into the thalamus promotes sleep in cats [39]. Glutamate also induces fictive locomotion in lamprey [40]. In these cases, however, the circuit mechanisms underlying glutamate's arousing effects are not known.

Comparing lethargus and adult arousal mechanisms
Mutants lacking NPR-1 exhibit accelerated locomotion in adults and during lethargus [11,18]. Several results suggest that locomotion arousal in adult and lethargus is established by a shared central sensory circuit. First, in both adult and lethargus, enhanced activity in the RMG sensory circuit accelerates locomotion, whereas decreased sensory transduction in the RMG circuit (i.e. by inactivating TAX-4 or OSM-9) abolishes npr-1's hyperactive locomotion defect [11,14], suggesting that the RMG circuit activity stimulates arousal in both awake and quiescent states. Second, EAT-4 acts in ASH and touch neurons to mediate hyperactive locomotion of npr-1 adult and lethargus stage animals, suggesting that glutamate release from these sensory neurons is required for locomotion arousal in npr-1 mutants.
On the other hand, several results suggest that the mechanisms that arouse locomotion differ between adult and lethargus animals. Inactivating GLR-2 AMPA receptors blocks the hyperactive locomotion of npr-1 mutants during lethargus but not in adults. Aroused locomotion in npr-1 adults persists in glr-1, glr-2, and nmr-1 mutants, indicating that other glutamate receptors are responsible for arousing adult locomotion. Similarly, artificial activation of ASH accelerates adult but not lethargus locomotion. Collectively, our results suggest that multiple sensory circuits govern locomotion arousal throughout development but that the relative contribution of each circuit to arousal differs depending on the developmental stage.

Materials and Methods Strains
Strain maintenance and genetic manipulation were performed as described [41]. Animals were cultivated at 20°C on agar nematode growth media (NGM) seeded with OP50 (for imaging and behavior) or HB101 E.coli (for electrophysiology). Wild type reference strain was N2 Bristol. Strains used in this study are as follows: 5' regulatory sequence: ASK expression), or mec-4 (~1.1kb 5' regulatory sequence: Touch neuron expression) promoters.

Transgenes and germline transformation
Transgenic strains were generated by microinjection of various plasmids with coinjection markers (myo-2p::NLS-mCherry (KP#1480) and vha-6p::mcherry (KP#1874)). Injection concentration was 40-50 ng/μl for all the expression constructs and 10 ng/μl for coinjection markers. The empty vector pBluescript was used to bring the final DNA concentration to 100 ng/μl. The flp-21 promoter (which is expressed in the RMG, ASH, ADL, ASK, URX, and ASI neurons [14]) was used to express transgenes in the RMG circuit.

Lethargus locomotion and behavior analysis
Lethargus locomotion was analyzed as previously described [11]. Well-fed late L4 animals were transferred to full lawn OP50 bacterial plates. After 1 hour, locomotion of animals in lethargus (determined by absence of pharyngeal pumping) was recorded on a Zeiss Discovery Stereomicroscope using Axiovision software. Locomotion was recorded at 2 Hz for 60 seconds. Centroid velocity of each animal was analyzed at each frame using object-tracking software in Axiovision. Motile fraction of each animal was calculated by dividing the number of frames with positive velocity value with total number of frames. Speed of each animal was calculated by averaging the velocity value at each frame. Quantitative analysis was done using a custom written MATLAB program (Mathworks). Statistical significance was determined using one-way ANOVA with Tukey test for multiple comparisons and two-tailed Student's t test for pairwise comparison.

Adult locomotion and behavior analysis
Locomotion of adult animals was analyzed with the same setup as lethargus locomotion analysis described above, except that well-fed adult animals were monitored 1-1.5hr after the transfer to full lawn OP50 bacterial plates. For the capsaicin treatment (Fig 4I), 1 day-old animals were transferred to NGM plates containing 50 μM capsaicin (with food), treated with capsaicin for 5 hours, and recorded for their locomotion. Statistical significance was determined using one-way ANOVA with Tukey test for multiple comparisons and two-tailed Student's t test for pairwise comparison.

Aldicarb assay
Sensitivity to aldicarb was determined by analyzing the time course of paralysis following treatment with 1 mM aldicarb (Sigma-Aldrich) as previously described [42]. Briefly, movement of animals was assessed by prodding animals with a platinum wire every 10 minute following exposure to aldicarb. 20-30 animals were tested for each trial. For the capsaicin treatment (Fig 4J), adult animals were transferred to NGM plates containing 50 μM capsaicin (with food), treated with capsaicin for 2-3 hours, and assayed for their paralysis on 1 mM aldicarb plates containing 50 μM capsaicin.

Electrophysiology
Electrophysiology was performed on dissected adult worms as previously described [43]. Worms were superfused in an extracellular solution containing 127 mM NaCl, 5 mM KCl, 26 mM NaHCO 3 , 1.25 mM NaH 2 PO 4 , 20 mM glucose, 1 mM CaCl 2 , and 4 mM MgCl 2 , bubbled with 5% CO 2 , 95% O 2 at 20°C. Whole cell recordings were carried out at -60 mV using an internal solution containing 105 mM CsCH 3 SO 3 , 10 mM CsCl, 15 mM CsF, 4mM MgCl 2 , 5mM EGTA, 0.25mM CaCl 2 , 10mM HEPES, and 4 mM Na 2 ATP, adjusted to pH 7.2 using CsOH. Under these conditions, we only observed endogenous acetylcholine EPSCs. To record GABAergic postsynaptic currents, the holding potential was 0 mV, at which we only observe mIPSCs. All recording conditions were as described [44]. To record evoked EPSCs, a 0.4 ms, 30 μA square pulse was applied to a motor neuron cell body with a stimulating electrode placed near the ventral nerve cord (one muscle distance from the recording pipette). Statistical significance was determined using one-way ANOVA with Tukey test for multiple comparisons and two-tailed Student's t test for pairwise comparison.

Fluorescence microscopy and image analysis
Quantitative imaging of coelomocyte fluorescence was performed as previously described [11] using a Zeiss Axioskop equipped with an Olympus PlanAPO 100x (NA = 1.4) objective and a CoolSNAP HQ CCD camera (Photometrics). Worms were immobilized with 30 mg/ml BDM (Sigma). The anterior coelomocytes were imaged in L4/A lethargus (determined by absence of pharyngeal pumping), and 1 day-old adult animals. Image stacks were captured and maximum intensity projections were obtained using Metamorph 7.1 software (Universal Imaging). YFP fluorescence was normalized to the absolute mean fluorescence of 0.5 mm FluoSphere beads (Molecular Probes). Statistical significance was determined using Kolmogorov-Smirnov test.

Calcium imaging and analysis
Using Dermabond topical skin adhesive, individual worms were glued to 2% agarose pads in extracellular saline (145 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 5 mM MgCl 2 , 20 mM D-glucose, and 10 mM HEPES buffer [pH7.2]). To image copper and glycerol responses, single animals were placed in a perfusion chamber (RC-26GLP,Warner Instruments) under a constant flow rate (0.4 ml min -1 ) of buffer using a perfusion pencil (AutoMate). Outflow was regulated using a peristaltic pump (Econo Pump, Bio-Rad). 10mM CuCl 2 (copper(II)chloride dihydrate, Sigma) or 500mM glycerol (Fisher) were delivered using the perfusion, pencil and switch between control and stimulus solutions was done using manually controlled valves. Solutions contained either 10mM CuCl 2 in M13 buffer or 500mM glycerol in 40mM NaCl, 1 mM MgSO 4 , 1 mM CaCl 2 and 5 mM KPO 4 . The stimulus was delivered for 10 seconds starting on the 10 th second from the beginning of the movie. Optical recordings were performed on a Zeiss Axioskop 2 upright compound microscope equipped with a Dual View beam splitter and a Uniblitz Shutter. Images were recorded at 10 Hz using an iXon EM camera (Andor Technology) and captured using IQ1.9 software (Andor Technology). For ratiometric imaging, ROI Y tracked the neuron in the yellow channel, and in the cyan channel, ROI C moved at a fixed offset from ROI Y . F was computed as F Y /F C following a correction for bleed through. No correction for bleaching was required. Ratio changes were detected and parametrized using scripts for MATLAB (The Mathworks). Briefly, the scripts average the F value for 5 preceding and including the marked start stimulus frame (F 0 ) and the 5 frames centered on the marked peak frame (F 1 ). ΔF was equal to (F 1 -F 0 ) / F 0 x 100. Touch-evoked calcium responses in PLM neurons were analyzed as previously described [11]. Statistical significance was determined using oneway ANOVA with Tukey test for multiple comparisons. worms has no effect on lethargus quiescence. Locomotion behavior of single worms during the L4/A lethargus (A-B) was analyzed in the indicated genotypes. Average motile fraction (A), and average locomotion velocity (B) are plotted. Transgenes that re-instated lethargus quiescence defects in eat-4;npr-1 (sra-6 or mec-4 promoted EAT-4, Fig 3) or glr-2;npr-1 (gcy-28d or nlp-12 promoted GLR-2, Fig 5) double mutants had no effect on lethargus quiescence in wild type worms. The number of animals analyzed is indicated for each genotype.