Natural sensory context drives diverse brain-wide activity during C. elegans mating.

Natural goal-directed behaviors often involve complex sequences of many stimulus-triggered components. Understanding how brain circuits organize such behaviors requires mapping the interactions between an animal, its environment, and its nervous system. Here, we use brain-wide neuronal imaging to study the full performance of mating by the C. elegans male. We show that as mating unfolds in a sequence of component behaviors, the brain operates similarly between instances of each component but distinctly between different components. When the full sensory and behavioral context is taken into account, unique roles emerge for each neuron. Functional correlations between neurons are not fixed but change with behavioral dynamics. From individual neurons to circuits, our study shows how diverse brain-wide dynamics emerge from the integration of sensory perception and motor actions in their natural context.

UBR-1 ubiquitin ligase regulates the balance between GABAergic and glutamatergic signaling.

Excitation/inhibition (E/I) balance is carefully maintained by the nervous system. The neurotransmitter GABA has been reported to be co-released with its sole precursor, the neurotransmitter glutamate. The genetic and circuitry mechanisms to establish the balance between GABAergic and glutamatergic signaling have not been fully elucidated. Caenorhabditis elegans DVB is an excitatory GABAergic motoneuron that drives the expulsion step in the defecation motor program. We show here that in addition to UNC-47, the vesicular GABA transporter, DVB also expresses EAT-4, a vesicular glutamate transporter. UBR-1, a conserved ubiquitin ligase, regulates DVB activity by suppressing a bidirectional inhibitory glutamate signaling. Loss of UBR-1 impairs DVB Ca2+ activity and expulsion frequency. These impairments are fully compensated by the knockdown of EAT-4 in DVB. Further, glutamate-gated chloride channels GLC-3 and GLC-2/4 receive DVB's glutamate signals to inhibit DVB and enteric muscle activity, respectively. These results implicate an intrinsic cellular mechanism that promotes the inherent asymmetric neural activity. We propose that elevated glutamate in ubr-1 mutants, being the cause of the E/I shift, potentially contributes to Johanson Blizzard syndrome.

The UBR-1 ubiquitin ligase regulates glutamate metabolism to generate coordinated motor pattern in Caenorhabditis elegans.

UBR1 is an E3 ubiquitin ligase best known for its ability to target protein degradation by the N-end rule. The physiological functions of UBR family proteins, however, remain not fully understood. We found that the functional loss of C. elegans UBR-1 leads to a specific motor deficit: when adult animals generate reversal movements, A-class motor neurons exhibit synchronized activation, preventing body bending. This motor deficit is rescued by removing GOT-1, a transaminase that converts aspartate to glutamate. Both UBR-1 and GOT-1 are expressed and critically required in premotor interneurons of the reversal motor circuit to regulate the motor pattern. ubr-1 and got-1 mutants exhibit elevated and decreased glutamate level, respectively. These results raise an intriguing possibility that UBR proteins regulate glutamate metabolism, which is critical for neuronal development and signaling.

Attenuation of insulin signalling contributes to FSN-1-mediated regulation of synapse development.

A neuronal F-box protein FSN-1 regulates Caenorhabditis elegans neuromuscular junction development by negatively regulating DLK-mediated MAPK signalling. In the present study, we show that attenuation of insulin/IGF signalling also contributes to FSN-1-dependent synaptic development and function. The aberrant synapse morphology and synaptic transmission in fsn-1 mutants are partially and specifically rescued by reducing insulin/IGF-signalling activity in postsynaptic muscles, as well as by reducing the activity of EGL-3, a prohormone convertase that processes agonistic insulin/IGF ligands INS-4 and INS-6, in neurons. FSN-1 interacts with, and potentiates the ubiquitination of EGL-3 in vitro, and reduces the EGL-3 level in vivo. We propose that FSN-1 may negatively regulate insulin/IGF signalling, in part, through EGL-3-dependent insulin-like ligand processing.

A Caenorhabditis elegans developmental decision requires insulin signaling-mediated neuron-intestine communication.

Adverse environmental conditions trigger C. elegans larvae to activate an alternative developmental program, termed dauer diapause, which renders them stress resistant. High-level insulin signaling prevents constitutive dauer formation. However, it is not fully understood how animals assess conditions to choose the optimal developmental program. Here, we show that insulin-like peptide (ILP)-mediated neuron-intestine communication plays a role in this developmental decision. Consistent with, and extending, previous findings, we show that the simultaneous removal of INS-4, INS-6 and DAF-28 leads to fully penetrant constitutive dauer formation, whereas the removal of INS-1 and INS-18 significantly inhibits constitutive dauer formation. These ligands are processed by the proprotein convertases PC1/KPC-1 and/or PC2/EGL-3. The agonistic and antagonistic ligands are expressed by, and function in, neurons to prevent or promote dauer formation. By contrast, the insulin receptor DAF-2 and its effector, the FOXO transcription factor DAF-16, function solely in the intestine to regulate the decision to enter diapause. These results suggest that the nervous system normally establishes an agonistic ILP-dominant paradigm to inhibit intestinal DAF-16 activation and allow reproductive development. Under adverse conditions, a switch in the agonistic-antagonistic ILP balance activates intestinal DAF-16, which commits animals to diapause.

A tonically active master neuron modulates mutually exclusive motor states at two timescales.

Continuity of behaviors requires animals to make smooth transitions between mutually exclusive behavioral states. Neural principles that govern these transitions are not well understood. Caenorhabditis elegans spontaneously switch between two opposite motor states, forward and backward movement, a phenomenon thought to reflect the reciprocal inhibition between interneurons AVB and AVA. Here, we report that spontaneous locomotion and their corresponding motor circuits are not separately controlled. AVA and AVB are neither functionally equivalent nor strictly reciprocally inhibitory. AVA, but not AVB, maintains a depolarized membrane potential. While AVA phasically inhibits the forward promoting interneuron AVB at a fast timescale, it maintains a tonic, extrasynaptic excitation on AVB over the longer timescale. We propose that AVA, with tonic and phasic activity of opposite polarities on different timescales, acts as a master neuron to break the symmetry between the underlying forward and backward motor circuits. This master neuron model offers a parsimonious solution for sustained locomotion consisted of mutually exclusive motor states.

C. elegans STRADalpha and SAD cooperatively regulate neuronal polarity and synaptic organization.

Neurons are polarized cells with morphologically and functionally distinct axons and dendrites. The SAD kinases are crucial for establishing the axon-dendrite identity across species. Previous studies suggest that a tumour suppressor kinase, LKB1, in the presence of a pseudokinase, STRADalpha, initiates axonal differentiation and growth through activating the SAD kinases in vertebrate neurons. STRADalpha was implicated in the localization, stabilization and activation of LKB1 in various cell culture studies. Its in vivo functions, however, have not been examined. In our present study, we analyzed the neuronal phenotypes of the first loss-of-function mutants for STRADalpha and examined their genetic interactions with LKB1 and SAD in C. elegans. Unexpectedly, only the C. elegans STRADalpha, STRD-1, functions exclusively through the SAD kinase, SAD-1, to regulate neuronal polarity and synaptic organization. Moreover, STRD-1 tightly associates with SAD-1 to coordinate its synaptic localizations. By contrast, the C. elegans LKB1, PAR-4, also functions in an additional genetic pathway independently of SAD-1 and STRD-1 to regulate neuronal polarity. We propose that STRD-1 establishes neuronal polarity and organizes synaptic proteins in a complex with the SAD-1 kinase. Our findings suggest that instead of a single, linear genetic pathway, STRADalpha and LKB1 regulate neuronal development through multiple effectors that are shared in some cellular contexts but distinct in others.

Functional imaging and quantification of multineuronal olfactory responses in C. elegans.

Many animals perceive odorant molecules by collecting information from ensembles of olfactory neurons, where each neuron uses receptors that are tuned to recognize certain odorant molecules with different binding affinity. Olfactory systems are able, in principle, to detect and discriminate diverse odorants using combinatorial coding strategies. We have combined microfluidics and multineuronal imaging to study the ensemble-level olfactory representations at the sensory periphery of the nematode Caenorhabditis elegans. The collective activity of C. elegans chemosensory neurons reveals high-dimensional representations of olfactory information across a broad space of odorant molecules. We reveal diverse tuning properties and dose-response curves across chemosensory neurons and across odorants. We describe the unique contribution of each sensory neuron to an ensemble-level code for volatile odorants. We show that a natural stimuli, a set of nematode pheromones, are also encoded by the sensory ensemble. The integrated activity of the C. elegans chemosensory neurons contains sufficient information to robustly encode the intensity and identity of diverse chemical stimuli.

Extrasynaptic signaling enables an asymmetric juvenile motor circuit to produce symmetric undulation.

In many animals, there is a direct correspondence between the motor patterns that drive locomotion and the motor neuron innervation. For example, the adult C. elegans moves with symmetric and alternating dorsal-ventral bending waves arising from symmetric motor neuron input onto the dorsal and ventral muscles. In contrast to the adult, the C. elegans motor circuit at the juvenile larval stage has asymmetric wiring between motor neurons and muscles but still generates adult-like bending waves with dorsal-ventral symmetry. We show that in the juvenile circuit, wiring between excitatory and inhibitory motor neurons coordinates the contraction of dorsal muscles with relaxation of ventral muscles, producing dorsal bends. However, ventral bending is not driven by analogous wiring. Instead, ventral muscles are excited uniformly by premotor interneurons through extrasynaptic signaling. Ventral bends occur in anti-phasic entrainment to activity of the same motor neurons that drive dorsal bends. During maturation, the juvenile motor circuit is replaced by two motor subcircuits that separately drive dorsal and ventral bending. Modeling reveals that the juvenile's immature motor circuit is an adequate solution to generate adult-like dorsal-ventral bending before the animal matures. Developmental rewiring between functionally degenerate circuit solutions, which both generate symmetric bending patterns, minimizes behavioral disruption across maturation.

Escape steering by cholecystokinin peptidergic signaling.

Escape is an evolutionarily conserved and essential avoidance response. Considered to be innate, most studies on escape responses focused on hard-wired circuits. We report here that a neuropeptide NLP-18 and its cholecystokinin receptor CKR-1 enable the escape circuit to execute a full omega (Ω) turn. We demonstrate in vivo NLP-18 is mainly secreted by the gustatory sensory neuron (ASI) to activate CKR-1 in the head motor neuron (SMD) and the turn-initiating interneuron (AIB). Removal of NLP-18 or CKR-1 or specific knockdown of CKR-1 in SMD or AIB neurons leads to shallower turns, hence less robust escape steering. Consistently, elevation of head motor neuron (SMD)'s Ca2+ transients during escape steering is attenuated upon the removal of NLP-18 or CKR-1. In vitro, synthetic NLP-18 directly evokes CKR-1-dependent currents in oocytes and CKR-1-dependent Ca2+ transients in SMD. Thus, cholecystokinin peptidergic signaling modulates an escape circuit to generate robust escape steering.

A putative cation channel, NCA-1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans.

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the alpha1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.

Corollary discharge promotes a sustained motor state in a neural circuit for navigation.

Animals exhibit behavioral and neural responses that persist on longer timescales than transient or fluctuating stimulus inputs. Here, we report that Caenorhabditis elegans uses feedback from the motor circuit to a sensory processing interneuron to sustain its motor state during thermotactic navigation. By imaging circuit activity in behaving animals, we show that a principal postsynaptic partner of the AFD thermosensory neuron, the AIY interneuron, encodes both temperature and motor state information. By optogenetic and genetic manipulation of this circuit, we demonstrate that the motor state representation in AIY is a corollary discharge signal. RIM, an interneuron that is connected with premotor interneurons, is required for this corollary discharge. Ablation of RIM eliminates the motor representation in AIY, allows thermosensory representations to reach downstream premotor interneurons, and reduces the animal's ability to sustain forward movements during thermotaxis. We propose that feedback from the motor circuit to the sensory processing circuit underlies a positive feedback mechanism to generate persistent neural activity and sustained behavioral patterns in a sensorimotor transformation.

Caenorhabditis elegans innexins regulate active zone differentiation.

In a genetic screen for active zone defective mutants in Caenorhabditis elegans, we isolated a loss-of-function allele of unc-7, a gene encoding an innexin/pannexin family gap junction protein. Innexin UNC-7 regulates the size and distribution of active zones at C. elegans neuromuscular junctions. Loss-of-function mutations in another innexin, UNC-9, cause similar active zone defects as unc-7 mutants. In addition to presumptive gap junction localizations, both UNC-7 and UNC-9 are also localized perisynaptically throughout development and required in presynaptic neurons to regulate active zone differentiation. Our mosaic analyses, electron microscopy, as well as expression studies suggest a novel and likely nonjunctional role of specific innexins in active zone differentiation in addition to gap junction formations.

An SCF-like ubiquitin ligase complex that controls presynaptic differentiation.

During synapse formation, specialized subcellular structures develop at synaptic junctions in a tightly regulated fashion. Cross-signalling initiated by ephrins, Wnts and transforming growth factor-beta family members between presynaptic and postsynaptic termini are proposed to govern synapse formation. It is not well understood how multiple signals are integrated and regulated by developing synaptic termini to control synaptic differentiation. Here we report the identification of FSN-1, a novel F-box protein that is required in presynaptic neurons for the restriction and/or maturation of synapses in Caenorhabditis elegans. Many F-box proteins are target recognition subunits of SCF (Skp, Cullin, F-box) ubiquitin-ligase complexes. fsn-1 functions in the same pathway as rpm-1, a gene encoding a large protein with RING finger domains. FSN-1 physically associates with RPM-1 and the C. elegans homologues of SKP1 and Cullin to form a new type of SCF complex at presynaptic periactive zones. We provide evidence that T10H9.2, which encodes the C. elegans receptor tyrosine kinase ALK (anaplastic lymphoma kinase), may be a target or a downstream effector through which FSN-1 stabilizes synapse formation. This neuron-specific, SCF-like complex therefore provides a localized signal to attenuate presynaptic differentiation.

Flexible motor sequence generation during stereotyped escape responses.

Complex animal behaviors arise from a flexible combination of stereotyped motor primitives. Here we use the escape responses of the nematode Caenorhabditis elegans to study how a nervous system dynamically explores the action space. The initiation of the escape responses is predictable: the animal moves away from a potential threat, a mechanical or thermal stimulus. But the motor sequence and the timing that follow are variable. We report that a feedforward excitation between neurons encoding distinct motor states underlies robust motor sequence generation, while mutual inhibition between these neurons controls the flexibility of timing in a motor sequence. Electrical synapses contribute to feedforward coupling whereas glutamatergic synapses contribute to inhibition. We conclude that C. elegans generates robust and flexible motor sequences by combining an excitatory coupling and a winner-take-all operation via mutual inhibition between motor modules.

An Upconversion Nanoparticle Enables Near Infrared-Optogenetic Manipulation of the Caenorhabditis elegans Motor Circuit.

Near-infrared (NIR) light penetrates tissue deeply, but its application to motor behavior stimulation has been limited by the lack of known genetic NIR light-responsive sensors. We designed and synthesized a Yb3+/Er3+/Ca2+-based lanthanide-doped upconversion nanoparticle (UCNP) that effectively converts 808 nm NIR light to green light emission. This UCNP is compatible with Chrimson, a cation channel activated by green light; as such, it can be used in the optogenetic manipulation of the motor behaviors of Caenorhabditis elegans. We show that this UCNP effectively activates Chrimson-expressing, inhibitory GABAergic motor neurons, leading to reduced action potential firing in the body wall muscle and resulting in locomotion inhibition. The UCNP also activates the excitatory glutamatergic DVC interneuron, leading to potentiated muscle action potential bursts and active reversal locomotion. Moreover, this UCNP exhibits negligible toxicity in neural development, growth, and reproduction, and the NIR energy required to elicit these behavioral and physiological responses does not activate the animal's temperature response. This study shows that UCNP provides a useful integrated optogenetic toolset, which may have wide applications in other experimental systems.

Excitatory motor neurons are local oscillators for backward locomotion.

Cell- or network-driven oscillators underlie motor rhythmicity. The identity of C. elegans oscillators remains unknown. Through cell ablation, electrophysiology, and calcium imaging, we show: (1) forward and backward locomotion is driven by different oscillators; (2) the cholinergic and excitatory A-class motor neurons exhibit intrinsic and oscillatory activity that is sufficient to drive backward locomotion in the absence of premotor interneurons; (3) the UNC-2 P/Q/N high-voltage-activated calcium current underlies A motor neuron's oscillation; (4) descending premotor interneurons AVA, via an evolutionarily conserved, mixed gap junction and chemical synapse configuration, exert state-dependent inhibition and potentiation of A motor neuron's intrinsic activity to regulate backward locomotion. Thus, motor neurons themselves derive rhythms, which are dually regulated by the descending interneurons to control the reversal motor state. These and previous findings exemplify compression: essential circuit properties are conserved but executed by fewer numbers and layers of neurons in a small locomotor network.

Human BAG-1 proteins bind to the cellular stress response protein GADD34 and interfere with GADD34 functions.

The cellular stress response protein GADD34 mediates growth arrest and apoptosis in response to DNA damage, negative growth signals, and protein malfolding. GADD34 binds to protein phosphatase PP1 and can attenuate the translational elongation of key transcriptional factors through dephosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha). Recently, we reported the involvement of human SNF5/INI1 (hSNF5/INI1) protein in the functions of GADD34 and showed that hSNF5/INI1 binds GADD34 and stimulates the bound PP1 phosphatase activity. To better understand the regulatory and functional mechanisms of GADD34, we undertook a yeast two-hybrid screen with full-length GADD34 as bait in order to identify additional protein partners of GADD34. We report here that human cochaperone protein BAG-1 interacts with GADD34 in vitro and in SW480 cells treated with the proteasome inhibitor z-LLL-B to induce apoptosis. Two other proteins, Hsp70/Hsc70 and PP1, associate reversibly with the GADD34-BAG-1 complex, and their dissociation is promoted by ATP. BAG-1 negatively modulates GADD34-bound PP1 activity, and the expression of BAG-1 isoforms can also mask GADD34-mediated inhibition of colony formation and suppression of transcription. Our findings suggest that BAG-1 may function to suppress the GADD34-mediated cellular stress response and support a role for BAG-1 in the survival of cells undergoing stress.

Altered p27(Kip1) phosphorylation, localization, and function in human epithelial cells resistant to transforming growth factor beta-mediated G(1) arrest.

p27(Kip1) is an important effector of G(1) arrest by transforming growth factor beta (TGF-beta). Investigations in a human mammary epithelial cell (HMEC) model, including cells that are sensitive (184(S)) and resistant (184A1L5(R)) to G(1) arrest by TGF-beta, revealed aberrant p27 regulation in the resistant cells. Cyclin E1-cyclin-dependent kinase 2 (cdk2) and cyclin A-cdk2 activities were increased, and p27-associated kinase activity was detected in 184A1L5(R) cells. p27 from 184A1L5(R) cells was localized to both nucleus and cytoplasm, showed an altered profile of phosphoisoforms, and had a reduced ability to bind and inhibit cyclin E1-cdk2 in vitro when compared to p27 from the sensitive 184(S) cells. In proliferating 184A1L5(R) cells, more p27 was associated with cyclin D1-cdk4 complexes than in 184(S). While TGF-beta inhibited the formation of cyclin D1-cdk4-p27 complexes in 184(S) cells, it did not inhibit the assembly of cyclin D1-cdk4-p27 complexes in the resistant 184A1L5(R) cells. p27 phosphorylation changed during cell cycle progression, with cyclin E1-bound p27 in G(0) showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G(1). These data suggest a model in which TGF-beta modulates p27 phosphorylation from its cyclin D1-bound assembly phosphoform to an alternate form that binds tightly to inhibit cyclin E1-cdk2. Altered phosphorylation of p27 in the resistant 184A1L5(R) cells may favor the binding of p27 to cyclin D1-cdk4 and prevent its accumulation in cyclin E1-cdk2 in response to TGF-beta.

Neuroendocrine modulation sustains the C. elegans forward motor state.

Neuromodulators shape neural circuit dynamics. Combining electron microscopy, genetics, transcriptome profiling, calcium imaging, and optogenetics, we discovered a peptidergic neuron that modulates C. elegans motor circuit dynamics. The Six/SO-family homeobox transcription factor UNC-39 governs lineage-specific neurogenesis to give rise to a neuron RID. RID bears the anatomic hallmarks of a specialized endocrine neuron: it harbors near-exclusive dense core vesicles that cluster periodically along the axon, and expresses multiple neuropeptides, including the FMRF-amide-related FLP-14. RID activity increases during forward movement. Ablating RID reduces the sustainability of forward movement, a phenotype partially recapitulated by removing FLP-14. Optogenetic depolarization of RID prolongs forward movement, an effect reduced in the absence of FLP-14. Together, these results establish the role of a neuroendocrine cell RID in sustaining a specific behavioral state in C. elegans.