Distribution and physiological effects of B-type allatostatins (myoinhibitory peptides, MIPs) in the stomatogastric nervous system of the crab Cancer borealis

Abstract

The crustacean stomatogastric ganglion (STG) is modulated by a large number of amines and neuropeptides that are found in descending pathways from anterior ganglia or reach the STG via the hemolymph. Among these are the allatostatin (AST) B types, also known as myoinhibitory peptides (MIPs). We used mass spectrometry to determine the sequences of nine members of the AST-B family of peptides that were found in the stomatogastric nervous system of the crab Cancer borealis. We raised an antibody against Cancer borealis allatostatin-B1 (CbAST-B1; VPNDWAHFRGSWa) and used it to map the distribution of CbAST-B1-like immunoreactivity (-LI) in the stomatogastric nervous system. CbAST-B1-LI was found in neurons and neuropil in the commissural ganglia (CoGs), in somata in the esophageal ganglion (OG), in fibers in the stomatogastric nerve (stn), and in neuropilar processes in the STG. CbAST-B1-LI was blocked by preincubation with 10(-6) M CbAST-B1 and was partially blocked by lower concentrations. Electrophysiological recordings of the effects of CbAST-B1, CbAST-B2, and CbAST-B3 on the pyloric rhythm of the STG showed that all three peptides inhibited the pyloric rhythm in a state-dependent manner. Specifically, all three peptides at 10(-8) M significantly decreased the frequency of the pyloric rhythm when the initial frequency of the pyloric rhythm was below 0.6 Hz. These data suggest important neuromodulatory roles for the CbAST-B family in the stomatogastric nervous system.

Figure 1

Direct tissue analysis of ganglia in the STNS. Mass spectra were obtained from a small piece of tissue from the STG (A) and CoG (B). Many peptides were detected, including B-type CbASTs (indicated with squares). C. Example of an MS/MS spectra for CbAST-B3 SGKWSNLRGAWa (m/z 1260.7) from direct tissue analysis of one STG; a, b and y ions are indicated.

Figure 2

CbAST-B1-like staining in the CoG. A. Maximum intensity projection. Immunoreactivity was seen in CoG cell bodies as well as in the neuropil. Immunoreactive fibers were apparent in the son and ion. Cell bodies and punctate labeling were often detected at the beginning of the son (boxed region). B. Mid-ganglion view of CbAST-B1-LI in the same ganglion as (A) emphasizing the presence of processes leading from the CoC into the CoG (asterisk). Fibers in the CoC also bypassed the CoG (arrowheads). Immunoreactive fibers in the son and ion (arrows) extended into the CoG neuropil. Fibers were also visible traveling between the brain and CoG neuropil in the CoC (double arrows). C. Another preparation, high magnification of fibers extending perpendicularly from the CoC fiber pathway to the CoG neuropil. Note in this case, two fibers can be seen extending to and branching out in the CoG neuropil. D. A cell and punctate staining in son (see boxed region in A).

Figure 3

CbAST-B1-like AST antibody staining in the OG and nerve backfills A-C. Neurobiotin backfill of the stn shows strong CbAST-B1-LI in both MPN somata (asterisks) and weak CbAST-B1-LI in the CD1 soma. Maximal intensity projections. D-F. Rhodamine-dextran backfill of the right ion labels 5 cells in the OG, of which one (asterisk) is strongly CbAST-B1-like immunoreactive. Maximal intensity projections.

Figure 4

CbAST-B1-LI in the anterior stn. A. Overview of the anterior stn and its junction with the two sons and the on. A large region of punctate staining is present in the anterior region of the stn. Fibers project from the two sons into the stn. Similar punctate staining was also detected in more posterior regions (arrow). B. High magnification of the anterior region of the stn from another preparation showing extensive punctate staining.

Figure 5

CbAST-B1 staining in the STG is restricted to the neuropil. STG somata were not immunoreactive while fibers in the neuropil stained extensively. Bulbous structures measuring up to ∼10 μm in length were detected in the central neuropil (arrowhead). Staining in the stn as it enters the STG is localized to one side of the nerve (single arrow). Fibers extended posteriorly into the dvn (double arrows).

Figure 6

Series of 2.5 μm-thick optical slices in a Z-stack of CbAST-B1-LI in the STG, with two dye-filled neurons. CbAST-B1-LI (magenta) was strongest around the periphery of the neuropil and virtually absent near the large diameter processes of the coarse, central neuropil. A VD cell was filled with Alexa Fluor 568 hydrazide (yellow), and a second, unidentified cell was filled with neurobiotin (green).

Figure 7

Summary of CbAST-B1-LI in the STNS of C. borealis.

Figure 8

CbAST-B1 and Dippu-AST 7 (A-type AST) immunoreactivity in the STNS. A. Maximal intensity projection of CbAST-B1-like staining in the CoG. Staining was detected in both cell bodies and neuropil. B. Maximal intensity projection of A-type AST staining in the same preparation. Arrows point to two large somata that were labeled. C. Overlay of A and B. D. Cell bodies in the CoG from the preparation in (C), showing some neurons colocalize the two families of peptides. Overlay of green and magenta in these somata appears white (blue asterisks). E. Maximal intensity projection of both CbAST-B1 (green) and Dippu-AST 7 (magenta) staining in the STG showing segregation of fiber staining in the stn and neuropil.

Figure 9

Physiological effects of CbAST-B1, CbAST-B2, and CbAST-B3 on the pyloric rhythm. A. Effects of 10^-5 M peptides on the pyloric rhythm. Representative intracellular recordings from the lateral pyloric (LP) neuron (top trace) and the corresponding extracellular recording from the lateral ventricular nerve (lvn, bottom trace), showing the activity of the PD (medium units), LP (largest unit), and PY (smallest unit) neurons. Application of either 10^-5 M CbAST-B1, 10^-5 M CbAST-B2 or 10^-5 M CbAST-B3 resulted in hyperpolarization of the LP neuron and silencing of the pyloric rhythm. B. Plot of initial pyloric burst frequency vs. final burst frequency for preparations exposed to either 10^-8 M CbAST-B1, 10^-8 M CbAST-B2, 10^-8 M CbAST-B3 plotted to show the burst frequency in the presence of the peptide as a function of the preparation's initial frequency (n=37). Preparations with a more rapid initial frequency responded less to peptide application that those with a slower initial burst frequency (Mann-Whitney Rank Sum Test, p=0.002).