The interneuron DVA and local search behavior

Prior work has demonstrated that specific classes of olfactory and gustatory neurons – in particular the olfactory neuron AWC – play central roles in monitoring the availability of food in the environment.^17,18 As noted above, sensory information about food availability is also transmitted through mechanosensory activation of dopaminergic neurons (CEP, ADE and PDE).¹⁹ What are the respective contributions of these sensory modalities to local food searching? Elegant Ca²⁺ imaging and behavioral studies have demonstrated that the AWC neuron responds robustly to odor removal, and promotes local searching behavior through synaptic contacts onto interneurons involved in the control of movement (AIA, AIB, AIY).^10,18,20 DVA makes synaptic contacts both onto interneurons that are primary postsynaptic partners of AIA, AIB and AIY, and onto downstream motor neurons (Fig. 1A). As noted above, DVA is strongly innervated by the dopaminergic neuron PDE. Mindful of this connectivity pattern, we proposed that changes in food availability may regulate NLP-12 release through dopaminergic inputs onto DVA from PDE. While direct dopaminergic regulation of DVA activity through synaptic release onto DVA is an appealing idea, it is also possible that other dopaminergic neurons influence DVA activity through an extrasynaptic mechanism. For example, the dopaminergic CEP neurons receive synaptic innervation from the ASK chemosensory neurons, and this could provide a parallel route for dopaminergic control. Control of NLP-12 release via dopaminergic regulation would provide a mechanism for conditional modulation of interneurons and motor neurons that play direct roles in controlling locomotory output. Due to strong synaptic innervation from the AIA, AIB and AIY neurons, the activity pattern of these motor circuit neurons is likely to also be heavily influenced by descending olfactory information about food availability. Thus, one interesting possibility is that olfactory and mechanosensory information about food availability converge at the level of premotor and motor neurons. Notably, recent work demonstrates the activity state of some premotor interneurons downstream of chemosensory neurons can have profound effects on neural responses to odors,²¹ providing an additional avenue by which peptide modulation of premotor and motor activity may shape local search responses.

If this is the case, it will be interesting to determine the temporal relationship between these sensory stimuli, motor circuit activity, and local search behavior. The AWC neuron is activated by odor removal, consistent with the notion that AWC activation may act as a trigger for initiation of local search behavior following removal from food.¹⁸ In contrast, dopaminergic neurons seem to be activated directly by the presence of food, making it difficult to understand how signaling through this pathway would modulate behavioral responses to removal from food. One possible explanation is that dopamine-mediated signaling events elicited by food exposure may not terminate immediately upon removal from food. In this case, the activation of dopaminergic neurons in the presence of food may produce a persistent elevation in DVA activity and NLP-12 release that extends beyond the duration of food exposure. Thus, mechanosensory information signaling recent food exposure may act in concert with olfactory information signaling food removal in order to shape local searching behavior.

DVA integration of sensory information

Previous work has suggested that DVA performs a proprioceptive function during locomotion.²² The transient receptor potential channel TRP-4 (homolog of mechanosensory TRPN channels) is expressed in DVA and deletion of trp-4 enhances body bend amplitude during forward locomotion. This effect is reversed by either DVA ablation or DVA-specific rescue of trp-4 expression. Further, manual bending of the worm was sufficient to produce trp-4-dependent Ca²⁺ increases in DVA. These results suggested that DVA is activated by stretch in order to modulate body bend depth. A subsequent study provided evidence that cholinergic synaptic release at the NMJ was potentiated by NLP-12 and also required trp-4, suggesting that DVA modulation of body bend amplitude may occur through NLP-12 potentiation of neuromuscular transmission.¹⁴

In our work we used a genetic strategy to increase body bend depth, and discovered a similar requirement for NLP-12 signaling. We engineered a mutation into muscle acetylcholine receptor (L-AChR, where L denotes levamisole-sensitive class) subunits that prolongs receptor activation and heightens synaptic activation of muscles in response to endogenous acetylcholine release. Expression of this gain of function receptor [L-AChR(gf)] produces a significant increase in body bend depth during movement (Fig. 2). We found that nlp-12 was required for these behavioral effects. Laser ablation of DVA reversed the exaggerated body bend phenotype of L-AChR(gf) expressing animals. In contrast, DVA ablation in wild type worms caused no obvious effect on movement, suggesting that DVA is recruited to modulate locomotion only under certain conditions, for example during the execution of deep body bends–a result consistent with previous evidence indicating a proprioceptive function for DVA. In support of this idea, the enhanced bending that was produced from NLP-12 overexpression was suppressed by a deletion mutation that eliminates L-AChR function in the muscles.

In the proper context: Neuropeptide regulation of behavioral transitions during food searching

All authors

Raja Bhattacharya & Michael M Francis

https://doi.org/10.1080/21624054.2015.1062971

Published online:

24 July 2015

Figure 2. Molecular signals in the control of body bend depth. Cartoon representing genetic manipulations that alter body bend depth. Transgenic expression of L-AChR(gf) receptors in muscles or DVA-specific overexpression of nlp-12 (nlp-12(OE)) increase body bend depth. Deletion of nlp-12 suppresses the locomotory effects of L-AChR(gf) expression. Conversely, mutation of the unc-29 AChR subunit (required for L-AChR function in muscles) suppresses the locomotory effects of nlp-12 overexpression. See text for additional details.

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Our findings using the L-AChR(gf) receptor provide support for a proprioceptive function for DVA, while our analysis of area-restricted search suggests context-dependent regulation of DVA activity based on food availability. Do these represent distinct functions of DVA? How might DVA distinguish between these sensory cues to appropriately shape behavior? We observed that wild type animals transiently increase their body bend depth following removal from food, perhaps suggesting that the proprioceptive and context-dependent functions of NLP-12 signaling are related. We propose that DVA integrates mechanosensory information encoded by dopaminergic neurons with cell-intrinsic information about stretch (via TRP-4 signaling) in order to shape locomotory output. In this case, the levels and timing of NLP-12 release would be regulated by the activity pattern of DVA and thereby alter behavioral responses accordingly.

A recent study reported an additional neuropeptide signaling system that resides in DVA and functions in sexually dimorphic manner.²³ Males deficient in the neuropeptide nematocin perform poorly in several steps of mating, including difficulty with turning. Either DVA ablation or DVA-specific disruption of the ntc-1 gene in males similarly disrupted mating behavior. Interestingly, other sensorimotor functions of ntc-1 mutant males were normal, consistent with a specific function of this peptide signaling system in the context of mating. Likewise, ntc-1 mutant hermaphrodites exhibited no discernable defects in locomotion. Thus, DVA may perform differing functions in the context of the specific behavioral state that is active, perhaps through selective release of peptide neuromodulators such as NLP-12 or NTC-1.

Mechanism of NLP-12 action

Most neuropeptides mediate their actions by activating specific G-protein coupled receptors (GPCR) that act through second messenger signaling pathways to alter neuronal properties over prolonged timescales compared with fast synaptic transmission. NLP-12 shares sequence similarity with mammalian gastrin/CCK peptides that have important peripheral roles in signaling satiety.²⁴ CCK is also abundant in the mammalian brain, though precise roles for CCK in modulation of brain circuits are only now beginning to emerge.²⁵ The effects of CCK in mammals are mediated through 2 GPCRs, CCK1 and CCK2, with distinct though partially overlapping distributions. CCK1 is abundant in peripheral tissues and is present in discrete brain areas, while CCK2 is predominant in the CNS.^26-28 In C. elegans, the Cholecystokinin-like Receptor 2 (CKR-2) GPCR was previously shown to bind synthetic NLP-12 peptides with high affinity in vitro.¹⁵ Moreover, nlp-12 and ckr-2 were similarly implicated in the regulation of fat storage, suggesting that this transmitter system plays conserved roles between nematodes and mammals.^15,29 Likewise, nlp-12 and ckr-2 were each required for short-term plasticity in cholinergic synaptic release at the NMJ.¹⁴ ckr-2 expression in cholinergic motor neurons was required for this effect, supporting the idea that NLP-12 acts via CKR-2 in cholinergic motor neurons to regulate their activity.

In contrast, we found that deletion of the ckr-2 gene did not impair food searching to the same degree as deletion of nlp-12, raising the interesting possibility that additional NLP-12 signaling pathways may function either independently or in parallel with CKR-2. Major postsynaptic targets of DVA include several command interneurons (AVE, AVA, AVB, PVC) as well as motor neurons (SMB, VA, VB and DB) that are directly involved in locomotion;¹⁶ thus, GPCRs expressed in these neurons might be prime candidates. In addition, NLP-12 might act in a hormonal manner through volume transmission, affecting GPCRs expressed on neurons that are not direct synaptic partners of DVA. Identifying the precise expression pattern of CKR-2 and any other GPCRs that bind NLP-12 will be essential for distinguishing between these modes of action, and for gaining a complete understanding of how NLP-12 regulates the local food search circuitry. For example, preferential activation of specific GPCR subtypes might underlie context-specific modulatory effects of NLP-12. One interesting possibility is that GPCRs which bind NLP-12 ligand with differing affinities might be differentially active depending upon levels of NLP-12 release.

In conclusion, our recent work in C. elegans has revealed a novel role for the NLP-12/CCK transmitter system in regulating behavioral responses to changes in food availability. Further, we demonstrated that NLP-12/CCK release is regulated by dopamine, and provided evidence that dopaminergic regulation of NLP-12 release plays a central role in shaping a context-dependent behavior, local food searching. Interestingly, similar interactions between dopamine and CCK have been described in mammalian neurons. Given the conserved nature of these signaling systems, we anticipate that ongoing investigation of this pathway will provide new insights into fundamental mechanisms that regulate feeding/foraging behaviors, and further, improve our understanding of general principles by which neuromodulators shape circuit activity and promote behavioral flexibility.