Identification of a new neuropeptide precursor reveals a novel source of extrinsic modulation in the feeding system of Aplysia

Abstract

The Aplysia feeding system is advantageous for investigating the role of neuropeptides in behavioral plasticity. One family of Aplysia neuropeptides is the myomodulins (MMs), originally purified from one of the feeding muscles, the accessory radula closer (ARC). However, two MMs, MMc and MMe, are not encoded on the only known MM gene. Here, we identify MM gene 2 (MMG2), which encodes MMc and MMe and four new neuropeptides. We use matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to verify that these novel MMG2-derived peptides (MMG2-DPs), as well as MMc and MMe, are synthesized from the precursor. Using antibodies against the MMG2-DPs, we demonstrate that neuronal processes that stain for MMG2-DPs are found in the buccal ganglion, which contains the feeding network, and in the buccal musculature including the ARC muscle. Surprisingly, however, no immunostaining is observed in buccal neurons including the ARC motoneurons. In situ hybridization reveals only few MMG2-expressing neurons that are mostly located in the pedal ganglion. Using immunohistochemical and electrophysiological techniques, we demonstrate that some of these pedal neurons project to the buccal ganglion and are the likely source of the MMG2-DP innervation of the feeding network and musculature. We show that the MMG2-DPs are bioactive both centrally and peripherally: they bias egestive feeding programs toward ingestive ones, and they modulate ARC muscle contractions. The multiple actions of the MMG2-DPs suggest that these peptides play a broad role in behavioral plasticity and that the pedal-buccal projection neurons that express them are a novel source of extrinsic modulation of the feeding system of Aplysia.

Figure 1.

MMG2 precursor protein. A signal peptide is shown by the white rectangle. Basic amino acids (K and R) are shown in bold. These are potential processing enzyme cleavage sites. Amidated peptides detected by MALDI (Fig. 4) are shown by the gray rectangles: MMc, MMe, and four novel peptides (MMG2-DPa, MMG2-DPb, MMG2-DPd, and MMG2-DPf). Peaks corresponding to the underlined nonamidated connecting peptides (pMMG2_84-112, 3091 Da; pMMG2_131-168, 4061 Da; pMMG2_171-199, 3231 Da) were also detected by MALDI (Fig. 4).

Figure 2.

Comparison of expression of MM and MMG2 mRNAs. Northern blots with either MM or MMG2 probes using RNA extracted from buccal (B), cerebral (C), pleural (Pl), pedal (Pe), and abdominal (A) ganglia are shown. Ribosomal RNA (rRNA) bands are shown to demonstrate that the same amount of RNA was loaded into each lane.

Figure 3.

ISH analysis of the expression of MMG2 in the CNS. Dorsal (A) and ventral (B) views of the central ganglia are shown. Scale bar, 100 μm. BG, Buccal ganglion; CG, cerebral ganglion; lPPG, left pleural and pedal ganglia; rPPG, right pleural and pedal ganglia; AG, abdominal ganglion.

Figure 4.

MALDI analysis of individual pedal neurons reveals MMG2 precursor processing. A representative mass spectrum of a single Aplysia pedal neuron is shown. The mass scale is divided into low (m/z 550-1150) (A), middle (m/z 1200-2250) (B), and high (m/z 2450-5500) (C) mass ranges. Mass spectral peaks were assigned based on the observed mass and the sequence of MMG2. D, Summary of the MMG2 precursor processing. Solid vertical lines represent cleavage sites, with black bars indicating dibasic sites that are entirely cleaved. Lightly shaded blocks represent peptides detected by MALDI. The processing of connecting peptide Asp¹²⁴-Tyr¹⁶⁸, highlighted below, shows numerous novel processing sites involving Leu-Leu and Leu-Ser cleavages.

Figure 5.

MMG2-DP immunostaining in the CNS. All panels are negatives of the black and white images collected at the appropriate filter settings. The inset above the ventral view of the buccal ganglion shows an enlarged view of the CBC illustrating an MMG2-DP-containing process. Scale bar, 100 μm. BG, Buccal ganglion; CG, cerebral ganglion; PPG, pleural and pedal ganglia; AG, abdominal ganglion; m, M-cluster; cbc, cerebro-buccal connective.

Figure 6.

MMG2-DP immunostaining in the ARC and I2 muscles. All panels are negatives of the black and white images collected at the appropriate filter settings. A, Staining of the ARC muscle with the MMG2-DPa antibody. B, Same field of view as in A stained with the buccalin antibody (Buc). C, Staining of the I2 muscle with the MMG2-DPa antibody. D, Same field of view as in C stained with the MMa antibody. The arrows in C and D point to a process stained with both the MMG2-DPa and MMa antibodies. Scale bar, 100 μm.

Figure 7.

Identification of MMG2-DP-containing neurons as pedal-buccal projection neurons (P-BPNs). A, Backfill of the CBC reveals several P-BPNs. B, Same ganglion stained for MMG2-DPs. C1, Carboxyfluoroscein injection of several neurons that projected to the CBC as verified by extracellular recording. C2, One representative recording of a P-BPN. Extracellular units recorded in the CBC correspond in a one-to-one manner to the spikes of the P-BPN. C3, Same ganglion as in C1 stained for MMG2-DPs. The asterisks indicate a MMG2-DP-containing neuron that did not project to the CBC. Scale bar: A, B, 500 μm; C1, C3, 300 μm.

Figure 8.

Effects of MMG2-DPb on ARC muscle contraction. A, MMG2-DPb induces baseline contracture of the muscle. The first and last phasic contractions elicited by bursts of motor neuron B15 firing are shown on an expanded time scale. MMG2-DPb (5 μm) was applied. B, The dose-response curve for the contracture induced by MMG2-DPb. Each point is the mean ± SE of measurements from three muscles. The smooth curve is a best-fit sigmoid function. C, The MMG2-DPb-induced contracture appears in an all-or-none, activity-dependent manner.

Figure 9.

Effects of the MMG2-DPs on esophageal nerve-elicited egestive motor programs. A, Representative recordings. Extracellular recordings of activity in the I2 nerve (I2N) and in buccal nerve 2 (Bn.2) and intracellular recordings of B8 and B4/5 firing were obtained simultaneously. The white bars mark the protraction phase, coincident with the activity in the I2 nerve. The black bars show the retraction phase, coincident with the activity in Bn.2. B, Group data of the effects of the MMG2-DPs on the B8 firing frequency in retraction, B4/5 firing frequency in retraction, and protraction duration. Error bars show the SE (n = 4-6). All ANOVAs were statistically significant (p < 0.05). Brackets show the groups compared in post hoc t tests. ^*p < 0.05, statistical significance. C, Dose dependence of the three effects of the MMG2-DPs. Data are the means ± SE from 4-10 preparations.

Figure 10.

MMG2-DPs decrease the excitability of B4/5. A, Representative recordings. B, Group data. The means ± SE are from eight preparations. Brackets show groups that were compared in two-tailed t tests after ANOVA. ^**p < 0.01, statistical significance.