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An important step in brain development is the remodeling of juvenile neural circuits to establish mature connectivity. The elimination of juvenile synapses is a critical step in this process; however, the molecular mechanisms directing synapse elimination activities and their timing are not fully understood. We identify here a conserved transcriptional regulator, DVE-1, that shares homology with mammalian special AT-rich sequence-binding (SATB) family members and directs the elimination of juvenile synaptic inputs onto remodelingC. elegansGABAergic neurons. Dorsally localized juvenile acetylcholine receptor clusters and apposing presynaptic sites are eliminated during maturation of wild type GABAergic neurons but persist into adulthood indve-1mutants. The persistence of juvenile synapses indve-1mutants does not impede synaptic growth during GABAergic remodeling and therefore produces heightened motor connectivity and a turning bias during movement. DVE-1 is localized to GABAergic nuclei prior to and during remodeling and DVE-1 nuclear localization is required for synapse elimination to proceed, consistent with DVE-1’s function as a transcriptional regulator. Pathway analysis of DVE-1 targets and proteasome inhibitor experiments implicate transcriptional control of the ubiquitin-proteasome system in synapse elimination. Together, our findings demonstrate a new role for a SATB family member in the control of synapse elimination during circuit remodeling through transcriptional regulation of ubiquitin-proteasome signaling.Contributions SummaryKDA generated strains, transgenic lines, molecular constructs, confocal microscopy images and analysis, performed optogenetic behavioral experiments, photoconversion experiments, modencode ChIP-seq analysis and pathway analysis. SR performed all calcium imaging experiments/analysis and conducted single worm tracking. KB performed all Bortezomib inhibitor experiments and analysis. CL generated most vectors and constructs. JR assisted with generation of CRISPR/Cas9 generated strains. WA and MR assisted with aldicarb behavioral assay. DO assisted with EMS screen and isolation ofdve-1mutant. CB and MD aided in CloudMap bioinformatic analysis of theuf171mutant. MMF and KDA designed and interpreted results of all experiments and wrote the manuscript.

The functional properties of neural circuits are defined by the patterns of synaptic connections between their partnering neurons, but the mechanisms that stabilize circuit connectivity are poorly understood. We systemically examined this question at synapses onto newly characterized dendritic spines of C. elegans GABAergic motor neurons. We show that the presynaptic adhesion protein neurexin/NRX-1 is required for stabilization of postsynaptic structure. We find that early postsynaptic developmental events proceed without a strict requirement for synaptic activity and are not disrupted by deletion of neurexin/nrx-1. However, in the absence of presynaptic NRX-1, dendritic spines and receptor clusters become destabilized and collapse prior to adulthood. We demonstrate that NRX-1 delivery to presynaptic terminals is dependent on kinesin-3/UNC-104 and show that ongoing UNC-104 function is required for postsynaptic maintenance in mature animals. By defining the dynamics and temporal order of synapse formation and maintenance events in vivo, we describe a mechanism for stabilizing mature circuit connectivity through neurexin-based adhesion.

SUMMARYNeuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here we show that the C. elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.Impact statementInvestigation of neuromodulatory control of ethologically conserved area-restricted food search behavior shows that NLP-12 stimulation of the head motor circuit promotes food searching through the previously uncharacterized CKR-1 GPCR.

In neural circuits, individual neurons often make projections onto multiple postsynaptic partners. Here, we investigate molecular mechanisms by which these divergent connections are generated, using dyadic synapses in C. elegans as a model. We report that C. elegans nrx-1/neurexin directs divergent connectivity through differential actions at synapses with partnering neurons and muscles. We show that cholinergic outputs onto neurons are, unexpectedly, located at previously undefined spine-like protrusions from GABAergic dendrites. Both these spine-like features and cholinergic receptor clustering are strikingly disrupted in the absence of nrx-1. Excitatory transmission onto GABAergic neurons, but not neuromuscular transmission, is also disrupted. Our data indicate that NRX-1 located at presynaptic sites specifically directs postsynaptic development in GABAergic neurons. Our findings provide evidence that individual neurons can direct differential patterns of connectivity with their post-synaptic partners through partner-specific utilization of synaptic organizers, offering a novel view into molecular control of divergent connectivity., eLife digest Nervous systems are complex networks of interconnected cells called neurons. These networks vary in size from a few hundred cells in worms, to tens of billions in the human brain. Within these networks, each individual neuron forms connections – called synapses – with many others. But these partner neurons are not necessarily alike. In fact, they may be different cell types. How neurons form distinct connections with different partner cells remains unclear. Part of the answer may lie in specialized proteins called cell adhesion molecules. These proteins occur on the cell surface and enable neurons to recognize one another. This helps ensure that the cells form appropriate connections via synapses. Cell adhesion molecules are therefore also known as synaptic organizers. Philbrook et al. have now examined the role of synaptic organizers in wiring up the nervous system of the nematode worm and model organism Caenorhabditis elegans. Motor neurons form connections with two types of partner cell: muscle cells and neurons. Philbrook et al. screened C. elegans that have mutations in genes encoding various synaptic organizers. This revealed that a protein called neurexin must be present for motor neurons to form synapses with other neurons. By contrast, neurexin is not required for the same neurons to establish synapses with muscles. Philbrook et al. found that neuron-to-neuron synapses arise at specialized finger-like projections. These resemble the dendritic spines at which synapses form in the brains of mammals, and had not been previously identified in C. elegans. In worms that lack neurexin, these spine-like structures do not form correctly, disrupting the formation of neuron-to-neuron connections. Previous work has implicated neurexin in synapse formation in the mammalian brain. But this is the first study to reveal a role for neurexin in establishing partner-specific synaptic connections. Mutations in synaptic organizers, including neurexin, contribute to disorders of brain development. These include schizophrenia and autism spectrum disorders. Learning more about how neurexin helps establish specific synaptic connections may help us understand how these disorders arise.

Establishing and maintaining the appropriate number of GABA synapses is key for balancing excitation and inhibition in the nervous system, though we have only a limited understanding of the mechanisms controlling GABA circuit connectivity. Here, we show that disrupting cholinergic innervation of GABAergic neurons in the C. elegans motor circuit alters GABAergic neuron synaptic connectivity. These changes are accompanied by a reduced frequency and increased amplitude of GABAergic synaptic events. Acute genetic disruption in early development–during the integration of post-embryonic born GABAergic neurons into the circuit–produces irreversible effects on GABAergic synaptic connectivity that mimic those produced by chronic manipulations. In contrast, acute genetic disruption of cholinergic signaling in the adult circuit does not reproduce these effects. Our findings reveal that GABAergic signaling is regulated by cholinergic neuronal activity, likely through distinct mechanisms in the developing and mature nervous system.

The circadian clock in the mammalian hypothalamic suprachiasmatic nucleus (SCN) is entrained by the ambient light/dark cycle, which differentially acts to cause the clock to advance or delay. Light-induced changes in the rhythmic expression of SCN clock genes are believed to be a critical step in this process, but how the two entrainment modalities—advances vs. delays—engage the molecular clockwork remains incompletely understood. We investigated molecular substrates of photic entrainment of the clock in the SCN by stably entraining hamsters to T cycles (non–24-h light/dark cycles) consisting of a single 1-h light pulse repeated as either a short (23.33-h) or a long (24.67-h) cycle; under these conditions, the light pulse of the short cycle acts as “dawn,” whereas that of the long cycle acts as “dusk.” Analyses of the expression of the photoinducible and rhythmic clock genes Period 1 and 2 ( Per1 and Per2 ) in the SCN revealed fundamental differences under these two entrainment modes. Light at dawn advanced the clock, advancing the onset of the Per1 mRNA rhythm and acutely increasing mRNA transcription, whereas light at dusk delayed the clock, delaying the offset of the Per2 mRNA rhythm and tonically increasing mRNA stability. The results suggest that the underlying molecular mechanisms of circadian entrainment differ with morning (advancing) or evening (delaying) light exposure, and such differences may reflect how entrainment takes place in nocturnal animals under natural conditions.

An organism's ability to thrive in changing environmental conditions requires the capacity for making flexible behavioral responses. Here we show that, in the nematode Caenorhabditis elegans, foraging responses to changes in food availability require nlp-12, a homolog of the mammalian neuropeptide cholecystokinin (CCK). nlp-12 expression is limited to a single interneuron (DVA) that is postsynaptic to dopaminergic neurons involved in food-sensing, and presynaptic to locomotory control neurons. NLP-12 release from DVA is regulated through the D1-like dopamine receptor DOP-1, and both nlp-12 and dop-1 are required for normal local food searching responses. nlp-12/CCK overexpression recapitulates characteristics of local food searching, and DVA ablation or mutations disrupting muscle acetylcholine receptor function attenuate these effects. Conversely, nlp-12 deletion reverses behavioral and functional changes associated with genetically enhanced muscle acetylcholine receptor activity. Thus, our data suggest that dopamine-mediated sensory information about food availability shapes foraging in a context-dependent manner through peptide modulation of locomotory output., Author Summary Animal behavior is profoundly affected by contextual information about the internal state of the organism as well as sensory information about the external environment. A class of signaling molecules known as neuropeptides have been implicated in driving transitions between behavioral states (e.g., from food seeking to satiety and back) but we have only a limited understanding of how neuropeptide signaling modulates neural circuit activity and elicits context-dependent behaviors. Here we identify a novel mechanism by which C. elegans modulate their behavior in response to sensory information about food. We show that dopaminergic regulation of NLP-12, a C. elegans homolog of the mammalian neuropeptide cholecystokinin (CCK), shapes behavioral transitions that are central to food searching. Given the conserved nature of these signaling pathways, our work raises the interesting possibility that dopamine modulation of CCK signaling represents a general mechanism by which nervous systems shape context-dependent behavioral changes.

Insulinoma-associated protein (IA)-2 and IA-2β are transmembrane proteins involved in neurotransmitter secretion. Mice with targeted disruption of both IA-2 and IA-2β (double-knockout, or DKO mice) have numerous endocrine and physiological disruptions, including disruption of circadian and diurnal rhythms. In the present study, we have assessed the impact of disruption of IA-2 and IA-2β on molecular rhythms in the brain and peripheral oscillators. We used in situ hybridization to assess molecular rhythms in the hypothalamic suprachiasmatic nuclei (SCN) of wild-type (WT) and DKO mice. The results indicate significant disruption of molecular rhythmicity in the SCN, which serves as the central pacemaker regulating circadian behavior. We also used quantitative PCR to assess gene expression rhythms in peripheral tissues of DKO, single-knockout, and WT mice. The results indicate significant attenuation of gene expression rhythms in several peripheral tissues of DKO mice but not in either single knockout. To distinguish whether this reduction in rhythmicity reflects defective oscillatory function in peripheral tissues or lack of entrainment of peripheral tissues, animals were injected with dexamethasone daily for 15 days, and then molecular rhythms were assessed throughout the day after discontinuation of injections. Dexamethasone injections improved gene expression rhythms in liver and heart of DKO mice. These results are consistent with the hypothesis that peripheral tissues of DKO mice have a functioning circadian clockwork, but rhythmicity is greatly reduced in the absence of robust, rhythmic physiological signals originating from the SCN. Thus, IA-2 and IA-2β play an important role in the regulation of circadian rhythms, likely through their participation in neurochemical communication among SCN neurons.

Circadian rhythms are rhythms in gene expression, metabolism, physiology, and behavior that persist in constant environmental conditions with a cycle length near 24 h. In mammals, the circadian timing system is hierarchical. The primary pacemaker regulating circadian behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Most cell types express circadian clock genes and will express rhythmicity in vitro. In vivo, the SCN entrains peripheral oscillators through a complex set of physiological and hormonal rhythms (31, 32, 36). At the molecular level, circadian oscillations are governed by a cell-autonomous negative-feedback loop in which transcription factors drive the expression of their own negative regulators, leading to oscillation between periods of transcriptional activation and repression (reviewed in references 32 and 36). The bHLH-PAS containing transcription factors CLOCK or NPAS2 form heterodimers with BMAL1. These heterodimers binds to E-box elements within regulatory regions of Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes to stimulate their transcription. Approximately 12 h after transcriptional activation, PER and CRY proteins reach concentrations sufficient to form repressor complexes that inhibit the activity of the CLOCK/NPAS2:BMAL1 heterodimer, reducing the transcription of Per and Cry genes and subsequently relieving PER/CRY-mediated negative feedback. E-box-mediated expression of other transcription factors, including members of the DBP/HLF/TEF and nuclear orphan receptor families (e.g., Rev-Erbα and ROR-A), provides a mechanism for clock control of genes with diverse promoters and with gene expression peaks occurring at a variety of phases. Posttranslational modifications of circadian clock proteins play a well-established role in the regulation of circadian cycle length. In both flies and mammals, phosphorylation of PER proteins by casein kinase 1 (CK1) proteins is thought to play a key step in determining the speed of the circadian clock (reviewed in reference 5). Mammalian cell culture studies indicate that the phosphorylation of PER proteins by CK1 epsilon (CK1ɛ) regulates their subcellular localization, likely affects their transcription repression capability, and promotes their degradation through a proteasomal pathway dependent upon the F-box proteins β-TrCP1 and β-TrCP2 (12, 29, 34, 35). Interference with β-TrCP1 activity lengthens circadian period in oscillating fibroblasts (27). The CK1 inhibitor IC261 and the proteasome inhibitors MG132 and lactacystin also lengthen period in fibroblasts (12). CRY proteins are also subjected to phosphorylation and degradation cycles that regulate circadian period. The F-box protein FBXL3 plays a key role in regulating CRY1 stability; mutations inactivating this gene increase circadian cycle length (6, 16, 30). Collectively, these studies indicate that the duration of activity of the PER/CRY repressor complex, regulated primarily by the stability of PER and CRY proteins, dictates the cycle length of the molecular oscillator (15). Genetic studies also support an important role for casein kinase action on PER proteins in regulating circadian period. A mutation in the Syrian hamster CK1ɛ gene, tau, shortens the circadian period of behavioral rhythms. Biochemically, the tau mutation (CK1ɛtau, a T178C substitution) differentially affects the activity of the kinase protein, reducing general kinase activity while increasing activity at specific residues of the PER proteins (14, 23). The tau mutation is a gain-of-function mutation with respect to circadian substrates, resulting in decreased PER stability and a reduction in circadian period length in tau mutant hamsters and mice (14, 24). In humans, familial advanced sleep phase syndrome (FASPS) is a circadian-based sleep disorder, in which affected individuals have a short circadian period and an advanced phase of the sleep-wake cycle. One study identified a FASPS pedigree with a mutation in human PER2 (hPER2; S662G mutation); this mutation prevents a priming phosphorylation, thus preventing CK1-mediated phosphorylation (33). A second study identified a dominant mutation within the kinase domain of CK1δ in a family with FASPS (38). Modeling this mutation in mice and flies revealed alterations in period length (38). In the circadian field, much of the attention on mammalian casein kinases has focused on CK1ɛ. The few studies examining CK1δ suggest that it plays a role similar to CK1ɛ. For example, CK1δ, like CK1ɛ, phosphorylates PER proteins, reducing their stability in vitro (7, 38), and both CK1δ and CK1ɛ are present in PER/CRY repressor complexes in vivo (21). Despite these similarities, the role of CK1δ in the molecular clockwork is not well understood. We report here results demonstrating important differences between CK1δ and CK1ɛ in the regulation of circadian cycle length, based on studies utilizing mice in which these genes have been inactivated.

Prokineticin 2 (PK2) is a putative output molecule from the SCN. PK2 RNA levels are rhythmic in the mouse SCN, with high levels during the day, and PK2 administration suppresses nocturnal locomotor activity in rats. The authors examined the PK2 system in a diurnal rodent, Arvicanthis niloticus, to determine whether PK2 or PK2 receptors differ between diurnal and nocturnal species. The major transcript variant of A. niloticus PK2 ( AnPK2) encodes a 26-residue signal peptide followed by the presumed mature peptide of 81 residues. Within the grass rat signal sequence, polymorphic sequences and amino acid substitutions were observed relative to mouse and laboratory rats, but the hydrophobic core and cleavage site of the signal sequence were preserved. The mature PK2 peptide is identical among A. niloticus, rat, and mouse. AnPK2 mRNA is rhythmically expressed in the SCN, with peak RNAlevels occurring in the morning, preceding peaks of Per1 and Per2 as in mouse SCN. Analysis of prokineticin receptor 2 (PKR2) sequences revealed polymorphisms among the grass rats studied. PKR2 mRNAwas expressed in the SCN and paraventricular nuclei of the thalamus and hypothalamus. While further analysis is necessary, there is no clear evidence indicating that a difference in the PK2 ligand/receptor system accounts for diurnality in this rodent species. These data contribute to a growing body of evidence suggesting that the key to diurnality lies downstream of the SCN in A. niloticus.

SummaryThe circadian clock mechanism in the mouse is composed of interlocking transcriptional feedback loops. Two transcription factors, CLOCK and BMAL1, are believed to be essential components of the circadian clock. We have used the Cre-LoxP system to generate whole-animal knockouts of CLOCK and evaluated the resultant circadian phenotypes. Surprisingly, CLOCK-deficient mice continue to express robust circadian rhythms in locomotor activity, although they do have altered responses to light. At the molecular and biochemical levels, clock gene mRNA and protein levels in both the master clock in the suprachiasmatic nuclei and a peripheral clock in the liver show alterations in the CLOCK-deficient animals, although the molecular feedback loops continue to function. Our data challenge a central feature of the current mammalian circadian clock model regarding the necessity of CLOCK:BMAL1 heterodimers for clock function.