A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior

Neuromodulators 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 Caenorhabditis 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

Identification and regulation of a novel nicotinic receptor at the Caenorhabditis elegans NMJ.

Identification and regulation of a novel nicotinic receptor at the Caenorhabditis elegans NMJ

Excitatory neurons sculpt GABAergic neuronal connectivity in the C. elegans motor circuit

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

A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior

SUMMARY Neuromodulators promote adaptive behaviors in response to either environmental or internal physiological changes. These responses are often complex and may involve concerted activity changes across circuits that are not physically connected. It is not well understood how neuromodulatory systems act across circuits to elicit complex behavioral responses. Here we show that the C. elegans NLP-12 neuropeptide system shapes responses to food availability by selectively modulating the activity of head and body wall motor neurons. NLP-12 modulation of the head and body wall motor circuits is generated through conditional involvement of alternate GPCR targets. The CKR-1 GPCR is highly expressed in the head motor circuit, and functions to enhance head bending and increase trajectory reorientations during local food searching, primarily through stimulatory actions on SMD head motor neurons. In contrast, NLP-12 activation of CKR-1 and CKR-2 GPCRs regulates body bending under basal conditions, primarily through actions on body wall motor neurons. Thus, locomotor responses to changing environmental conditions emerge from conditional NLP-12 stimulation of head or body wall motor neuron targets.status: publishe

The Anaphase-Promoting Complex (APC) ubiquitin ligase regulates GABA transmission at the C. elegans neuromuscular junction

Regulation of both excitatory and inhibitory synaptic transmission is critical for proper nervous system function. Aberrant synaptic signaling, including altered excitatory to inhibitory balance, is observed in numerous neurological diseases. The ubiquitin enzyme system controls the abundance of many synaptic proteins and thus plays a key role in regulating synaptic transmission. The Anaphase-Promoting Complex (APC) is a multi-subunit ubiquitin ligase that was originally discovered as a key regulator of protein turnover during the cell cycle. More recently, the APC has been shown to function in postmitotic neurons, where it regulates diverse processes such as synapse development and synaptic transmission at glutamatergic synapses. Here we report that the APC regulates synaptic GABA signaling by acting in motor neurons to control the balance of excitatory (acetylcholine) to inhibitory (GABA) transmission at the Caenorhabditis elegans neuromuscular junction (NMJ). Loss-of-function mutants in multiple APC subunits have increased muscle excitation at the NMJ; this phenotype is rescued by expression of the missing subunit in GABA neurons. Quantitative imaging and electrophysiological analyses indicate that APC mutants have decreased GABA release but normal cholinergic transmission. Consistent with this, APC mutants exhibit convulsions in a seizure assay sensitive to reductions in GABA signaling. Previous studies in other systems showed that the APC can negatively regulate the levels of the active zone protein SYD-2 Liprin-alpha. Similarly, we found that SYD-2 accumulates in APC mutants at GABAergic presynaptic sites. Finally, we found that the APC subunit EMB-27 CDC16 can localize to presynapses in GABA neurons. Together, our data suggest a model in which the APC acts at GABAergic presynapses to promote GABA release and inhibit muscle excitation. These findings are the first evidence that the APC regulates transmission at inhibitory synapses and have implications for understanding nervous system pathologies, such as epilepsy, that are characterized by misregulated GABA signaling

Locomotory phenotypes associated with L-AChR<i>(gf)</i> expression require neuropeptide signaling.

<p>(<b>A</b>) Average body bends/min measured in liquid for the genotypes indicated. The strong locomotory defects of <i>egl-3</i> and <i>egl-21</i> mutants prevented analysis of L-AChR(<i>gf</i>) effects on agar (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584.s003" target="_blank">Fig. S3A, B</a>). Mutation of <i>pkc-1</i> normalized the locomotor effects of L-AChR(<i>gf</i>) in both liquid and on agar (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584.s003" target="_blank">Fig. S3C–E</a>). (<b>B</b>) Movement trajectories of wild type, L-AChR<i>(gf), nlp-12</i>;L-AChR<i>(gf)</i> animals as indicated. Each black line shows the trajectory of one animal monitored for 45 s on food. (<b>C</b>) Average body bend amplitude for the genotypes indicated. Each bar represents the mean (±SEM) of values calculated from recordings of at least 15 animals. For (A) and (C) ***, p<0.0001 by ANOVA with Sidak's post-hoc test. (<b>D</b>) Wide-field epifluorescent image of an adult animal expressing <i>nlp-12::SL2::mCherry</i>. The image is oriented with the head to the left. White rectangle: nerve ring. Arrow: DVA cell body. (<b>E</b>) Average body bend amplitude for the indicated genotypes and effects of DVA ablation (–DVA). Ablations were performed on L2 stage animals. Body bend amplitude was measured from recordings of young adult animals 2 days following laser ablation. Error bars indicate mean (±SEM) of at least 8 animals. ***, p<0.0001 student's t-test.</p

Modulation of body bend depth through NLP-12/CCK signaling is critical for local food searching.

<p>(<b>A</b>) Schematic representation of the neural circuit underlying NLP-12 modulation of local searching. Synaptic connections (triangles, brackets) are as described by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Garrison1" target="_blank">[42]</a>. DVA receives synaptic input from the dopaminergic neuron PDE and makes connections with both motor neurons and interneurons involved in locomotory control. DVA makes synaptic contacts onto all of the motor and interneurons indicated by brackets. In addition, DVA is connected to AVB and PVC by gap junctions. Assignments of interneurons into layers are as described by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Gray1" target="_blank">[13]</a>. Other neuron classes are as described in the text and references therein. DOP-1 modulation of DVA activity regulates NLP-12 release from DVA, altering the motor pattern during local food searching. (<b>B</b>) Average body bend amplitude for the genotypes as indicated measured during an initial five minute time interval (0–5) immediately following removal from food and a second five minute time interval 30 minutes after removal from food (30–35). Bars represent mean values (±SEM) calculated from 9–14 animals. (<b>C</b>) Representative tracks of wild type and <i>nlp-12(ok335)</i> mutants during an initial five minute period (0–5) following removal from food. Note the decreased number of reorientations and increased frequency of long forward runs for <i>nlp-12(ok335)</i> mutant as compared to the wild type. (<b>D</b>) Total directional reorientations measured during the 0–5 and 30–35 minute intervals following removal from food for wild type, transgenic wild type animals expressing Tetanus toxin in DVA <i>[DVA::Tetx]</i>, <i>nlp-12(ok335)</i> and <i>ckr-2(tm3082)</i> mutants. Bars represent mean (±SEM) for at least 12 animals. (<b>E</b>) Quantification of reversal coupled omega turns and reorientations in the absence of omega turns during the first 5 minutes following removal from food for the genotypes indicated. (WT and <i>nlp-12(ok335)</i>: n = 17). Rescue refers to the <i>nlp-12(ok335)</i> mutant expressing an extrachromosomal array carrying <i>Pnlp-12::nlp-12::SL2::mCherry</i> (n = 9). Bars represent mean (±SEM). ***, p<0.0001; **, p<0.001 by ANOVA with Sidak's post-hoc test.</p

The dopamine receptor DOP-1 is required in DVA for NLP-12 modulation of food searching.

<p>(<b>A</b>) Frequency of high angled reorientations for wild type and <i>nlp-12(ok335)</i> animals quantified for 5 minutes after transfer to food free plates in the presence (+) or absence (−) of dopamine (DA). Bars represent mean (±SEM) for at least 12 animals. Dopamine mechanosensory signaling is strongly enhanced at low osmotic strength <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Schafer1" target="_blank">[73]</a>. Therefore, these assays were conducted following transfer of the animals to low osmotic-strength assay plates as described previously <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004584#pgen.1004584-Hills1" target="_blank">[14]</a>. We observed a modest increase in basal reorientation frequency across all genotypes under these conditions. (<b>B, C</b>) Representative images (B) and quantification (C) of NLP-12::VenusYFP fluorescence in the ventral cord region of the DVA process of wild type, <i>dop-1(vs100)</i>, and <i>dop-1(vs100) Ex DVA::dop-1</i> animals before (−) and after (+) 10 minutes dopamine (DA) treatment (wild type: n = 12 for (−) and (+) DA; <i>dop-1(vs100)</i>: n = 12 for (−) and 9 for (+) DA). <i>Ex DVA::dop-1</i> refers to specific rescue of <i>dop-1</i> expression in DVA using the <i>nlp-12</i> promoter (−DA, n = 12; +DA, n = 11). Bars represent mean ±SEM. ***, p<0.0005; *, p<0.05 student's t-test. (<b>D</b>) Single slice confocal images of the DVA neuron in a transgenic animal expressing <i>nlp-12::SL2::mCherry</i> (upper panel) together with <i>Pdop-1::GFP</i> (middle panel). White arrow denotes the DVA interneuron in all cases. Asterix denotes a ventral cord motor neuron expressing the <i>dop-1</i> reporter. Scale bars in B and D, 20 µm. (<b>E</b>) Total directional reorientations measured during 0–5 and 30–35 minute intervals following removal from food for the genotypes as indicated. WT: n = 10; <i>dop-1(vs101)</i>: n = 12, <i>dop-1(vs100)</i>: n = 14, <i>dop-1(vs100) Ex DVA::dop-1</i>: n = 12 and <i>dop-3(vs106)</i>: n = 8. Bars represent mean (±SEM). For (A) and (E) ***, p<0.0005, **, p<0.005 by ANOVA with Sidak's post-hoc test.</p