Functional characterization of a neuropeptide receptor exogenously expressed in Aplysia neurons

Neuropeptides act mostly on a class of G-protein coupled receptors, and play a fundamental role in the functions of neural circuits underlying behaviors. However, the functions of neuropeptide receptors are poorly understood. Here, we used the mollusc model system Aplysia and microinjected the exogenous neuropeptide receptor apATRPR (Aplysia Allatotropin-related peptide receptor) with an expression vector (pNEX3) into Aplysia neurons that did not express the receptor. Physiological experiments demonstrated that apATRPR could mediate the excitability increase activated by its ligand, apATRP (Aplysia Allatotropin-related peptide), in the Aplysia neurons that now express the receptor. This study provides the first definitive evidence for a physiological function of a neuropeptide receptor in molluscan animals.


Introduction
Neuropeptides are the most diverse class of neurotransmitters/neuromodulators, which mostly act on G-protein coupled receptors (GPCRs). The diversity arises in part from the possibility that a single neuropeptide precursor can generate multiple forms of active peptides, and a peptide can act on multiple GPCRs, which in turn might function through different signaling pathways [1,2].
Recently, growing genetic information in model systems is becoming available and has facilitated studying of both neuropeptides and their receptors [36,[48][49][50][51][52]. In the latter studies [49,50], a common approach is to express putative GPCRs in a cell line, and test activity of potential ligands on the receptor. Then the receptor expression in the CNS and physiological and/or circuit activity of the ligands are demonstrated. If the receptor activity of the ligands in the cell line matches their physiological activity in the CNS, it is used as evidence that the receptor functions in the CNS.
However, given that a peptide might act on multiple receptors, it is necessary to demonstrate that the identified GPCR actually displays the proper physiological activity in native neurons. In this paper, we have used an expression vector [3][4][5], to develop a method that expresses a peptide GPCR in native Aplysia neurons, and examine whether the GPCR could show a physiological activity. Our research utilizes Aplysia allatotropin (apATRP) [39] and its receptor apATRPR [50] as an example.
The neuropeptide allatotropin was first found in tissues of corpora allata in the insect Manduca Sexta, and it stimulated the secretion of juvenile hormone [53]. Subsequently, Allatotropin-related 4 peptides have been characterized in animals across phyla, including Arthropoda [54], Annelida [48] and Mollusca [39] with multiple functions in different behaviors, including feeding. The allatotropin receptor was originally characterized in Bombyx mori [55], followed by more insect allatotropin receptors, such as in Aedes aegypti [56] and Tribolium castaneum [57]. Additionally, two allatotropin receptors in the annelid Platynereis [48] and one in Aplysia [50] were characterized.
Interestingly, although allatotropin in the protostomes and orexin/hypocretin in vertebrates/deuterostomes [58] display no obvious similarity other than the amidated C-terminal, recent work has shown that their receptors are orthologous to each other based on phylogenetic analyses, supporting the evolutionary homology of allatotropin and orexin signaling systems [59,60].
In Aplysia, apATRP (GFRLNSASRVAHGY-NH2) functions in the feeding network have been extensively characterized. apATRP targets B61/B62 motor neurons in the buccal ganglia by enhancing their excitability [39]. B61/B62 is a site of plasticity after learning that food is inedible [26]. Recently, several ligands, including apATRP, were found to activate apATRPR in CHO-K1 cells transiently transfected with apATRPR. Importantly, the pattern of activations of these ligands in the cell line matches their actions on B61/62 excitability, suggesting that apATRPR likely functions in the Aplysia CNS [50]. However, it is unknown whether apATRPR mediates the excitability increase in native Aplysia neurons. This is a critical question, given that there might be multiple apATRP receptors in Aplysia. Here, we sought to determine whether apATRP is sufficient to mediate the ligand effect on native neurons by evaluating the ability of apATRP to activate apATRPR in Aplysia neurons that do not originally express apATRPR.
To express apATRPR in neurons, we used a plasmid vector, pNEX (plasmid for neuronal expression), which is a reliable and effective method to express exogenous proteins in cultured Aplysia neurons [5,61]. Initially, pNEX was constructed with the AK01a gene (shaker K + channel), and microinjected into Aplysia neurons, which demonstrated that the AK01a channel could modulate the firing of the injected neuron and regulate synaptic interactions [61]. Subsequent studies used pNEX to overexpress the target proteins and elucidated molecular mechanisms of synaptic plasticity [62][63][64][65][66][67][68]. Notably, small molecule neurotransmitter receptors (1ɑ metabotropic glutamate receptor [69]) and biogenic amine receptors (octopamine receptor) [64]), which are G-protein coupled receptors, were also expressed in Aplysia neurons through the vector pNEX. However, to date, no studies have applied this technique to neuropeptide GPCRs. Here, we used pNEX3 to successfully express apATRPR in Aplysia neurons that originally did not respond to ATRP. After expression, the neurons showed excitability increase in response to apATRP, indicating that apATRPR can mediate the excitability increase.
The plasmid pNEX3-EGFP was derived from the earlier work [73], and the plasmid pcDNA3.1-apATRPR was a gift from Dr. Checco at the University of Illinois. To generate the expression plasmid pNEX3-apATRPR, the apATRPR gene was ligated with the vector pNEX3. First, the EGFP gene was digested from the BamHI-KpnI restriction fragment of pNEX3 and separated by agarose 6 gel electrophoresis. Second, the apATRPR gene was added to the restriction sites of BamHI and KpnI at the 5' and 3' ends (forward primer: CGCGGATCCATGGGGTCGAACGATACATTC; reverse primer: GGGGTACCTCAGATGCTGGCGAGAGTGACCTC), respectively, by performing polymerase chain reaction (PCR) with the pcDNA3.1-apATRPR plasmid as the template. Then, the target gene, apATRPR, and the vector, pNEX3, were ligated using T4 DNA ligase. All plasmid DNAs used in microinjection were prepared by a standard maxi-prep procedure using an EndoFree Maxi Plasmid Kit.
Aplysia are hermaphroditic (i.e., each animal has functioning male and female reproductive organs).
Animals were kept in an aquarium containing aerated and filtered artificial seawater (Instant Ocean, Aquarium Systems Inc., Mentor, OH) at 14−16 °C. The animal room was equipped with a 24 h light−dark cycle with a light period from 6:00 am to 6:00 pm. Prior to dissection, animals were anesthetized by injection of isotonic 333 mM MgCl 2 (approximately 50% of body weight) into the body cavity. All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. apATRP was synthesized by ChinaPeptides Co., Ltd.
Briefly, ganglia were desheathed, transferred to a recording chamber containing 1.

Microinjection of plasmids
For neurons in the Aplysia buccal ganglion that didn't respond to apATRP, we established two groups. The control neurons were microinjected with the DNA construct pNEX3-EGFP, and the experimental neurons were microinjected with a mixture of pNEX3-EGFP and pNEX3-apATRPR.
The EGFP was used as a marker of gene expression. We microinjected the plasmids by pressure injection. The pressure ranged from 20 to 30 psi. To observe the microinjected plasmid volume, we mixed the plasmids with 2% fast green buffer (20 mM HEPES, 200 mM KCl, pH = 7.37) at 1:1. We stopped injecting when the plasmid volume expanded 1/3 of the volume of the injected neuron and the neuron could be seen to turn green due to fast green (see Fig 1B). In the experimental group, if neurons had green fluorescence under an Olympus fluorescence microscope (see Fig 1C), we assumed that these neurons expressed both EGFP and apATRPR.

Culture of Aplysia neurons and detection of plasmid expression
8 After injection, the buccal ganglia were cultured at 18 °C. The culture medium was made up of an aliquot of Aplysia hemolymph and L15 at 1:1 by volume [79,80]. Then, we added a 1% total volume of 50 mg/ml ampicillin sodium salt (Sigma-Aldrich: A9518-25G-9) solution and a 1% total volume of 200 mM L-glutamine (Sigma-Aldrich: V900419-100G). The culture medium was prepared The mixture was filter-sterilized through a 0.22 μm filter, and stored at 4℃.
During ganglia culture, the gene expression was observed using an Olympus fluorescence microscope every day. If the neurons in the control and experimental groups expressed green fluorescence, we then evaluated the activity of apATRP in these neurons using the procedures described in the "Electrophysiology" section.

Construction of plasmid pNEX3-ATRPR
The recombinant plasmid, pNEX3-apATRPR, was identified by digestion and the DNA sequencing ( Fig 1A). The verified recombinant plasmid was used to microinject Aplysia neurons in the experimental group. The plasmid pNEX3-EGFP was microinjected into neurons in the control group.

apATRP has no effect on neurons B1/B2
To demonstrate that apATRPR might function as an endogenous receptor of apATRP, we sought to find a target neuron that did not natively express apATRPR in the buccal ganglia. We selected a larger neuron, B8 (~ 150 μm), and examined B8 excitability changes in response to apATRP. apATRP could increase B8 excitability (Fig 2 A-B, F(3, 6)

Microinjection of pNEX3-apATRPR into the target neuron
In each hemi-ganglion of the buccal ganglion, there are one B1 and one B2 neuron. Thus, there are four B1/B2 neurons on both sides of the buccal ganglion. We set up two groups: the plasmid pNEX3-EGFP mixture with fast green microinjected into B1/B2 neurons as the control group, and the plasmid pNEX3-EGFP and pNEX3-apATRPR mixture with fast green microinjected into the contralateral B1/B2 neurons as the experimental group. Fast green can be visualized with a regular light source in a microscope, which allowed us to make sure that the plasmid injection was successful ( Fig 1B).
After injection, we placed the buccal ganglion into the cell culture for 1-3 days, and observed whether B1/B2 neurons exhibited green fluorescence. If we observed green fluorescence, we considered that neurons were successful in expressing the EGFP in the control group, and coexpressing the apATRPR and EGFP in the experimental group. In 32 cells injected with these plasmids, 30% of the cells subsequently expressed the genes.

Discussion
In this work, we have characterized physiological functions of a neuropeptide receptor, apATRPR, expressed in Aplysia neurons. Earlier [39,50], we showed that apATRPR is expressed in the Aplysia CNS. Moreover, apATRP and several other ligands can activate apATRPR expressed in a cell line, and these effects matched their enhancing effects on B61/B62excitability. These pieces of evidence are consistent with the notion that apATRPR may mediate the effects of ATRP on B61/B62 neurons.
However, prior to the present work, no direct evidence showed that apATRPR could mediate excitability increase in an Aplysia neuron. This evidence is critical given two considerations. First, apATRPR activity in a cell line is often measured by a ligand's ability to increase IP1 concentration in Gαq signaling pathway, whereas excitability increase likely requires an ultimate action of a GPCR on some specific ionic channels [81]. Second, there might be additional ATRP receptor(s) other than the identified apATRPR in Aplysia that might mediate excitability increase. Indeed, two allatotropin receptors have been characterized in annelid Platynereis [48], which together with mollusc Aplysia, belongs to a superphylum: lophotrochozoa.
To provide further evidence, we sought to determine if apATRPR might mediate excitability increase in a neuron that does not express apATRPR. Among several buccal neurons, B1/B2 neurons did not show an excitability increase in response to apATRP. To express exogenous genes in Aplysia neurons, we used the plasmid vector pNEX3 [5, 67-69, 75, 76] to construct pNEX3-EGFP and pNEX3-apATRPR, and microinjected B1/B2 neurons in the control group with pNEX3-EGFP, those in the experimental group with both pNEX3-EGFP and pNEX3-apATRPR. B1/B2 neurons in the experimental group could be excited by apATRP, whereas B1/B2 neurons in the control group could not. Thus, our study provides the first evidence that apATRPR indeed mediates excitability increase in a neuron that does not express apATRPR. Taken together with earlier work showing that pNEXδ or pNEX3 can express GPCRs for glutamate or octopamine [64,69], pNEX, including pNEXδ and pNEX3, proves to be an effective plasmid to express GPCRs for both small molecule transmitters and neuropeptides in Aplysia neurons.
We expect that such a procedure could be readily applied to demonstrate physiological functions of neuropeptide receptors in native neurons in model systems with reasonably large identifiable neurons, such as other molluscs, annelids and possibly some arthropods. Notably, compared with invertebrate genetic organisms C elegans [82] and Drosophila [83], life spans of molluscs and annelids are relatively long, making it difficult to use transgenes to manipulate gene expression. 13 Consequently, the procedure described in this paper should be particularly useful in these animals to study functions of genes in native neurons.
In summary, we provide direct evidence indicating that the neuropeptide receptor, apATRPR, is sufficient to mediate an excitability increase to its ligand, apATRP, in Aplysia neurons. This is a further proof that Aplysia is an advantageous model system for the study of peptidergic neuromodulation in well-defined neural circuits for feeding, locomotion and other behaviors.

Acknowledgements
Not applicable.  were made in high divalent saline. Bars in A and C denote current injections in B1/B2.