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1Department of Neuroscience, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; 2Department of Chemistry and School of Pharmacy, University of Wisconsin–Madison, Madison, Wisconsin; and 3Laboratory of Developmental Physiology, Genomics and Proteomics, Katholieke Universiteit Leuven, Leuven, Belgium
Submitted 25 July 2006; accepted in final form 22 October 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Marder et al. 2005). Neuromodulators gain access to these motor networks by neuronal and/or neuroendocrine release. This neuronal release often occurs from the terminals of projection neurons or sensory inputs (Beenhakker and Nusbaum 2004; Billimoria et al. 2005; Blitz et al. 2004; Christie et al. 2004; Koh and Weiss 2005; Proekt et al. 2005). Ultimately, to fully understand motor network operation in the unrestrained animal, it will be necessary to identify and characterize all the neuromodulators that have access to that network. Thus far, only a subset of the modulatory inputs has been so studied in any single model system.
One small motor system where a full characterization of all sources of neuromodulation is potentially achievable is the stomatogastric nervous system (STNS) of the crab Cancer borealis (Marder and Bucher 2006; Nusbaum and Beenhakker 2002). Many modulatory influences to this system are identified, with each one having distinct actions (Marder and Bucher 2006; Nusbaum et al. 2001). The STNS, which consists of four ganglia plus their connecting and peripheral nerves, contains a set of interacting central pattern generating (CPG) circuits underlying various aspects of feeding. The two best characterized of these CPGs, the gastric mill (chewing) and pyloric (filtering) circuits, are located in the stomatogastric ganglion (STG). These CPGs are modulated by projection neuron inputs originating in the neighboring, paired commissural ganglia (CoGs) and unpaired esophageal ganglion (OG) (Marder and Bucher 2006; Nusbaum et al. 2001). They also receive hormonal modulation from several sources, including the paired pericardial organs (POs) (Marder et al. 2005).
Herein we report the identity and influence of pyrokinin (PK) peptides in the C. borealis STG. The PK peptide family occurs in the central and peripheral nervous systems of many arthropods and its actions on peripheral tissues are well studied (Choi and Jurenka 2004; Choi et al. 2001; Clynen et al. 2003; Holman et al. 1986; Predel et al. 1997; Schoofs et al. 1991; Torfs et al. 2001; Veelaert et al. 1997; Zdárek et al. 2002). However, its actions have yet to be determined in the CNS of any species. Here we localize and identify the amino acid sequences of Cancer borealis pyrokinins I and II (CabPK-I, CabPK-II) and show that these PKs excite all gastric mill circuit neurons and elicit the gastric mill rhythm in the isolated STG. Whereas many previously identified modulators elicit and modify the pyloric rhythm, this is the first neuromodulator in C. borealis whose application to the isolated STG activates the gastric mill rhythm. The CabPK-elicited gastric mill rhythm occurs in the absence of activity in modulatory commissural neuron 1 (MCN1), a projection neuron that also elicits the gastric mill rhythm. When driving the gastric mill rhythm, MCN1 is also a CPG neuron for this rhythm (Coleman and Nusbaum 1994; Coleman et al. 1995). These results therefore indicate that the CPGs underlying these two gastric mill rhythms are distinct.
Some of these data were previously published in abstract form (Hertzberg et al. 2003).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Male Jonah crabs (Cancer borealis) were obtained from Commercial Lobster and Seafood (Boston, MA) and the Marine Biological Laboratory (Woods Hole, MA). Before experimentation, the crabs were housed in commercial tanks containing recirculating, aerated artificial seawater (10°C). Before dissection, the crabs were cold-anesthetized by packing them in ice for 
30 min. The foregut was then removed and maintained in chilled physiological saline while the STNS was dissected away from the foregut.
Solutions
The STNS was maintained in physiological saline containing (in mM): 440 NaCl, 26 MgCl2, 13 CaCl2, 11 KCl, 10 Trizma base, and 5 maleic acid (pH 7.4–7.6). CabPK-I and CabPK-II (Biotechnology Center, University of Wisconsin, Madison, WI), Pevpyrokinin-2 (PPK-2: ADFAFSPRLamide; Protein Chemistry Laboratory, University of Pennsylvania School of Medicine, Philadelphia, PA), and Leucopyrokinin (LPK: pETSFTPRLamide; Bachem, King of Prussia, PA; Protein Chemistry Laboratory, University of Pennsylvania School of Medicine, Philadelphia, PA) were prepared in physiological saline immediately before use. Picrotoxin (PTX: 10–5 M; Sigma–Aldrich, St. Louis, MO) was prepared in physiological saline, with stirring for 40 min, and superfused to suppress glutamatergic inhibitory synaptic transmission in the STG (Marder 1987; Marder and Paupardin-Tritsch 1978). PTX suppresses the synaptic actions of most gastric mill neurons, including those of the reciprocally inhibitory lateral gastric (LG) neuron and interneuron 1 (Int1) (Marder and Paupardin-Tritsch 1978). Int1 and the LG neuron are key gastric mill CPG neurons (Bartos et al. 1999; Coleman et al. 1995).
Antiserum development
Synthetic PPK-1 (DFAFSPRLamide) was coupled, through the free carboxyl group of its N-terminal aspartate residue, to bovine thyroglobulin using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC; Sigma, St. Louis, MO). This procedure increased the likelihood that the resulting antiserum generated against thyroglobulin-bound PPK-1 would be directed against the C-terminal portion of the PK peptide, which is conserved across the different members of this peptide family.
After overnight incubation, the water-soluble isourea, which is released as a by-product of the conjugation reaction, and the excess reagent were separated from the hapten-carrier complex by dialysis. The complex was then dissolved in distilled water and emulsified with an equal amount of Freunds complete adjuvant and injected subcutaneously into New Zealand white rabbits. Second, third, and fourth boosts were given, using Freunds incomplete adjuvant, 2, 4, and 6 wk, respectively, after the initial immunization.
Dot immunobinding assay
The antiserum obtained after the final bleed was characterized using dot immunobinding assay (DIA) according to Salzet et al. (1997). Briefly, 1 µl of each of several synthetic peptides was spotted onto a nitrocellulose membrane (0.45 µm pore size) in a dilution series ranging from 100 pg/dot to 
1 µg/dot. These peptides included PPK-1, PPK-2, Pev-kinin-6 (AFSPWAamide; Torfs et al. 1999), C. borealis tachykinin-related peptide-Ia (CabTRP-Ia: APSGFLGMRamide; Christie et al. 1997; Nieto et al. 1998), and Pev-Sulfakinin-2 [Pev-SK 2: pQFDEY(SO3H)GHMRFamide; Torfs et al. 2002]. Membranes were baked for 30 min at 110°C, blocked with skimmed milk in 50 mM Tris-buffered saline (TBS) to reduce background staining (1 h gentle agitation at room temperature), and subsequently incubated overnight with the primary antiserum in a dilution of 1/1,000 in TBS. Membranes were washed several times and incubated with horseradish peroxidase–linked goat anti-rabbit IgG at 1/500 dilution for 2 h at ambient temperature. Immunoreactive spots were visualized by incubation with a 0.3% 3,3' diamino-benzidine (DAB)/H2O2 solution.
As reported in Torfs (2001), DIA analysis of the anti-PPK-1 antiserum readily stained PPK-1 peptide at peptide levels down to 1 ng/dot and stained PPK-2 peptide at levels down to 0.1 µg/dot (lowest level tested). In contrast, this antiserum did not stain dots containing Pev-Kinin-6, CabTRP-Ia, or Pev-SK 2 at levels 1 µg/dot (Torfs 2001). Tissue sections were also processed using the anti-PPK-1A antiserum, at a dilution of 1/1,000, with labeling visualized with the peroxidase antiperoxidase (PAP) method (VandeSande and Dierickx 1976). As earlier, DAB was used as the chromogenic agent.
Tissue preparation
The CNS (brain, subesophageal ganglion, thoracic ganglia, and ventral nerve cord) of the adult white shrimp Penaeus vannamei (obtained from the Centro Nacional de Acuicultura e Investigaciones Marinas, Guayaquil, Ecuador) was dissected under physiological saline and transferred to Bouins Hollande’s (10%) sublimate fixative. After 18–24 h of fixation, the CNS was rinsed with distilled water (12 h), dehydrated in an ethanol series (70, 95, and 100% for two times 1 h each), cleared in Histosol plus, and embedded in Paraplast. Alternating sections of 4 µm were made with a LKB Historange microtome using glass knives.
Whole-mount immunocytochemistry
Whole-mount immunocytochemistry was performed using standard techniques for the C. borealis STNS (Blitz et al. 1999; Christie et al. 2004; Saideman et al. 2006). Briefly, tissue was fixed for 4–24 h with either 4% paraformaldehyde or 4% EDAC in 0.1 M sodium phosphate (P: pH 7.3) or Bouin’s fixative (71% saturated picric acid, 24% of 40% formalin, 5% glacial acetic acid). Preparations were then rinsed five times at 1-h intervals in 0.1 M P with 0.3% Triton X-100 (P-Triton, pH 7.3). Tissue was subsequently incubated with primary antiserum and diluted to final concentration with P-Triton, for 24–48 h. PK-like immunoreactivity (PK-LI) was examined with the polyclonal anti-PPK antiserum (anti-PPK-1A) at a final dilution of 1:300 to 1:500. The tissue was then rinsed as above with P-Triton and incubated for 15–24 h with goat anti-rabbit secondary antisera conjugated to Alexa Fluor 488 or Alexa Fluor 546 (Molecular Probes, Eugene, OR). The secondary antisera were used at a final dilution of 1:300 in P-Triton. After incubation with secondary antisera, preparations were rinsed five times at 1-h intervals with P. They were then mounted between a glass slide and coverslip, using a solution of 80% glycerol and 20% 20 mM Na2CO3 (pH 9.0). Digital images were taken with a Leica DMRB microscope and Leica DC 350FX digital camera system (Leica Microsystems, Bannockburn, IL). Images were acquired with Image-Pro Express software (Media Cybernetics, Silver Spring, MD).
Preadsorption control experiments were conducted to confirm the specificity of anti-PK immunolabeling. The polyclonal antibody (1:500) was preincubated for 4 h at room temperature with PPK (10–5 M) or an unrelated neuropeptide, corazonin (10–4 M: pQTFQYSRGWTNamide; Bachem, King of Prussia, PA). Corazonin localizes to the POs in C. borealis (Li et al. 2003). In some additional preparations, we omitted either the preadsorbing peptide or the primary antiserum but otherwise processed the whole mounts as above for preadsorption controls.
Tissue extraction
Supraesophageal ganglion (brain) tissue and CoGs were pooled from nearly 100 animals (C. borealis) and two separate extractions were performed on each pool of ganglia. Tissues were placed in a 0.1- or 1.0-ml tissue grinder (Wheaton, Millville, NJ) with 100 µl acidified methanol (90/9/1, methanol/glacial acetic acid/deionized water, vol/vol/vol) storage buffer. The tissue was homogenized completely, after which the extraction liquid was transferred to a 1.5-ml microcentrifuge tube (Fisher Scientific, Pittsburgh, PA) and centrifuged at 13,200 x g for 10 min in an Eppendorf 5415 D microcentrifuge (Brinkmann Instruments, Westbury, NY). The supernatant was retained and the resulting pellet reextracted and respun. Supernatants were combined and concentrated to near dryness with a Savant SC 110 SpeedVac concentrator (Thermo Electron, West Palm Beach, FL). Finally, a minimal amount (100–150 µl) of resuspension solution (deionized water with 0.1% formic acid) was added to the extract. This resuspended extract was vortexed and briefly centrifuged, with the supernatant used for high-performance liquid chromatography (HPLC) separation followed by immuno-dot-blot assay and matrix-assisted laser desorption/ionization (MALDI)–Fourier transform mass spectrometry (FTMS) screening or liquid chromatography tandem mass spectrometry (LC MS/MS) analysis of the immunopositive HPLC fractions.
Reverse-phase HPLC separations
HPLC separations were performed using a Rainin Dynamax HPLC system equipped with a Dynamax UV-D II absorbance detector (Rainin Instrument, Woburn, MA). The mobile phases used include: (Solution A) deionized water containing 0.1% formic acid and (Solution B) acetonitrile (HPLC grade, Fisher Scientific) containing 0.1% formic acid. Twenty microliters of extract were injected onto a Macrosphere C18 column (2.1 mm ID x 250 mm length, 5-µm particle size; Alltech Associates, Deerfield, IL). Two HPLC experiments were performed on aliquots of the extract. The first separation was a 60-min gradient of 5%-95% Solution B. The second was a shallower gradient across 120 min, which allowed a higher-resolution separation. To obtain more peptide in each fraction and to facilitate identification, the second gradient was performed on six separate injections of brain extract and fractions were pooled for a final volume of 1.2 ml. Fractions were automatically collected every minute using a Rainin Dynamax FC-4 fraction collector.
Immuno-dot-blot assay
The HPLC fractions were assayed for immunoreactivity to anti-PPK antiserum. We used 30 µl from each fraction and added 6 µl of 1 mg/ml bovine serum albumin (BSA, RIA grade; Fluka, Buchs, Switzerland) in PBS, after which each fraction was concentrated to dryness in a SpeedVac. Each dried fraction was resuspended in 6 µl of deionized water, spotted in 1-µl aliquots onto a nitrocellulose membrane (Schleicher & Schuell Bioscience, Keene, NH), and dried in a 60°C oven for 1 h. The membrane was then fixed for 16 h at 37°C with 2% glutaraldehyde (EM grade; Ted Pella, Redding, CA) vapor in a sealed container. A 2% glutaraldehyde solution in PBS was subsequently added to the membrane and incubated for 1 h to complete the fixation. The blots were washed six times for 15 min each in PBS, blocked with blotto (5% nonfat dry milk, 0.1% Triton X-100, 0.001% Thimerosal in PBS) for 30 min, and then incubated with PPK antiserum diluted 1:500,000 in blotto for 16 h at 4°C. A 10% blotto solution was used, three times for 15 min, to rinse unbound antibody from the blots. Secondary goat anti-rabbit antiserum coupled to horseradish peroxidase at a dilution of 1:500 in blotto was then incubated with the blot for 1 h. Again the unbound antibodies were removed by three rinses in 10% blotto for 15 min. Bound antibodies were visualized by adding the blot to a solution of 0.006% H2O2, 0.03% 3,3'-diaminobenzidine 4-HCl, 0.5% CoCl2 in PBS for 4–5 min. The membrane was washed thoroughly in water and dried at room temperature. Immunoreactive fractions were identified by a reddish-brown staining at the location at which the fraction had been added to the membrane (Sithigorngul et al. 1991, 2003).
Direct tissue sample preparation
For direct tissue mass spectrometric analysis, samples were prepared as described by Kutz et al. (2004). Briefly, dissected and desheathed tissue was rinsed in acidified methanol. Tissue was desalted in dilute MALDI matrix consisting of 10 mg/ml 2,5-dihydroxybenzoic acid (DHB; MP Biomedicals, Irvine, CA) prepared in deionized water. A spot containing 0.3 µl saturated DHB [150 mg/ml in 50/50 vol/vol water/purge trap-grade methanol (Fisher Scientific)] was placed on one facet of the IonSpec MALDI sample probe (IonSpec, Lake Forest, CA). Before the DHB crystallized fully, the tissue was placed carefully on the facet and an additional 0.3-µl drop of saturated DHB was placed on top of the tissue to help affix the tissue to the target. The saturated DHB was allowed to crystallize at room temperature.
MALDI FTMS
MALDI FTMS experiments were performed on a Fourier transform mass spectrometer (IonSpec) equipped with a 7.0-Tesla actively shielded superconducting magnet. The FTMS instrument contained an external ion source using a quadrupole ion guide to transfer the ions to the ion cyclotron resonance (ICR) cell, which was differentially pumped. The sample probe was a ten-faceted stainless steel target. A 337-nm nitrogen laser (Laser Science, Franklin, MA) was used for ionization/desorption. All mass spectra were collected in positive ion mode using the in-cell accumulation method. The latter method was written using IonSpec version 7.0 software, as described previously (Kutz et al. 2004). The pulse sequence involved the use of seven laser ionization/desorption events, each being optimized for ions at m/z 1,000 to be transported down the quadrupole. This method was used to increase the ion concentration before detection. Five pulse sequences were signal averaged to further improve the signal-to-noise ratio. The instrument was externally calibrated using Substance P and several matrix ions. Additionally, the mass spectra were internally calibrated to known neuropeptides.
Capillary LC-ESI-QTOF-MS/MS
Nanoscale LC QTOF MS/MS was performed using a Waters capillary LC system coupled to a quadrupole time-of-flight (QTOF) micromass spectrometer (Waters, Milford, MA). Chromatographic separations were performed on a C18 reverse-phase capillary column (75 µm ID x 150 mm length, 3-µm particle size; Micro-Tech Scientific). The mobile phases used were: (Solution A) deionized H2O with 5% acetonitrile and 0.1% formic acid; (Solution B) acetonitrile with 5% deionized H2O and 0.1% formic acid; and (Solution C) deionized H2O with 0.1% formic acid. A 1.4-µl aliquot of PK immunopositive C. borealis brain HPLC fraction was injected and loaded onto the trap column (PepMap C18; 300 µm column internal diameter x 1 mm, 5-µm particle size; LC Packings, Sunnyvale, CA) using mobile phase C at a flow rate of 30 µl/min for 3 min. After this procedure, the stream select module was switched to a position at which the trap column became in-line with the analytical capillary column and a linear gradient of mobile phases A and B was initiated. A splitter was added between the mobile phase mixer and the stream select module to reduce the flow rate from 12 µl/min to 200 nl/min.
The nanoflow electrospray ionization (ESI) source conditions were set as follows: capillary voltage, 3,800 V; sample cone voltage, 40 V; extraction cone voltage, 1 V; source temperature, 120°C; cone gas (N2) 13 L/h. For the reference spray, the same settings were used except that the sample cone voltage was set at 10 V and reference scans were performed every 10 s. A data-dependent acquisition was used for the MS survey scan and the selection of precursor ions and subsequent MS/MS of the selected parent ions. The MS scan range was from m/z 100 to m/z 2,000 and the MS/MS scan was from m/z 50 to m/z 2,000. The MS/MS de novo sequencing was performed with a combination of manual sequencing and automatic sequencing by PepSeq software (Waters).
Electrophysiology
Electrophysiological experiments were performed using standard techniques for this system (Beenhakker and Nusbaum 2004). The isolated STNS was pinned down in a Sylgard 184–lined (KR Anderson, Santa Clara, CA) petri dish (Fig. 1). All preparations were superfused continuously (7–12 ml/min) with C. borealis physiological saline (10–12°C). Each extracellular recording was made by pressing a stainless steel wire electrode into the Sylgard alongside the nerve and isolating that area with petroleum jelly (Vaseline: Lab Safety Supply, Janesville, WI). The second electrode was connected to ground by insertion into the Sylgard in the main bath compartment. To facilitate intracellular recordings, the desheathed ganglia were viewed with light transmitted through a dark-field condenser (Nikon, Tokyo, Japan). Intracellular recordings of STG somata were made using microelectrodes (15–30 M
) filled with 4 M K-acetate plus 20 mM KCl or 0.6 M K2SO4 plus 10 mM KCl. Intracellular current injection was performed using Axoclamp 2 amplifiers (Molecular Dynamics, Foster City, CA) in single-electrode discontinuous current-clamp (DCC) mode. Sample rates during DCC were 2–3 kHz.
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). Data were collected directly onto a chart recorder (Model MT-95000 or Everest; Astromed, West Warwick, RI) and simultaneously digitized (about 5 kHz) and collected onto a PC computer using data acquisition/analysis tools (Spike2, Cambridge Electronic Design, Cambridge, UK). To study neuromodulatory inputs in isolation, the CoGs were cut away by bisecting the inferior esophageal nerves (ions) and superior esophageal nerves (sons) to remove modulatory input from the CoG projection neurons. The axon of each MCN1, which projects through the ipsilateral ion, was stimulated extracellularly (tonic stimulation: 4–10 Hz) using a Grass Telefactor S88 stimulator, by a stimulus isolation unit (SIU5: Astro-Med/Grass Telefactor, West Warwick, RI). In most experiments, CabPK-I, CabPK-II, PPK, or LPK was superfused continuously across the isolated STG at 10–6 M concentration. These four PK peptides had identical actions so, except where noted, the data resulting from their application were pooled.
Data analysis was facilitated with a custom-written program for Spike2 that determines the activity levels and phase relationships of neurons (freely available at http://www.neurobiologie.de/). Unless otherwise stated, each datum in a data set was derived by determining the average of 10 consecutive impulse bursts. Briefly, burst duration was defined as the duration between the onset of the first and last action potential in an impulse burst. The firing rate of a neuron was defined as the number of action potentials minus 1, divided by the burst duration. The cycle frequency of the pyloric rhythm was determined by calculating the inverse of the period between the onsets of two successive pyloric dilator (PD) neuron bursts. Gastric mill cycle period was determined as the duration between successive impulse bursts in the LG neuron.
Figures were made from Spike2 files incorporated into Adobe Illustrator (Adobe, San Jose, CA) and Powerpoint graphics programs (Microsoft, Seattle, WA). Statistical analyses (two-tailed Student’s t-test or one-way repeated-measures ANOVA) were performed with SigmaStat 3.0 and SigmaPlot 8.0 (SPSS, Chicago, IL). Data are expressed as means ± SD.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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We identified PK-LI in the C. borealis STNS (Fig. 1). Specifically, there consistently was PK-LI in the STG neuropil but not in any STG somata (n = 25) (Fig. 1). There was also PK-LI in what appeared to be four or five individual axons that entered the STG from the stomatogastric nerve (stn), but there was no PK-LI in any peripheral nerves projecting from the STG (Fig. 1). The PK-LI within the STG neuropil thus appeared to originate from projection neurons in neighboring ganglia. In C. borealis, nearly all the projection neurons that innervate the STG originate in the CoGs (Coleman et al. 1992).
Consistent with the possibility that PK-LI–containing projection neurons in the CoGs innervated the STG, there was also PK-LI in about two axons within each son and in a subset of neuronal somata in each CoG (n = 21) (Fig. 1). As many as 20 PK-LI somata were counted in individual CoGs (mean: 8 ± 4 somata, n = 20) and, consistently, there was PK-LI in the CoG neuropil. No PK-LI was observed in either the ions or the OG (n = 27). Finally, there was also PK-LI in the neuropil region at the anterior end of the stn, near its junction with the sons and esophageal nerve (n = 20) (Fig. 1).
PK-LI was also found in the POs, a major neurohemal site that releases neuromodulators into the pericardial cavity (Fu et al. 2005; Skiebe 2001). The PK-LI labeling was sparsely distributed in axon fibers and varicosities throughout the POs (not shown). This immunolabeling occurred consistently across preparations (n = 12/15).
To increase the likelihood that the PK-LI represented immunolabeling of antibody binding to PK peptides, we performed preadsorption controls. Specifically, preincubating the PPK antiserum with PPK (10–5 M) abolished PK-LI in STNS whole-mount preparations (n = 5). Omitting the primary antiserum also abolished labeling (n = 7). In contrast, whole mounts in which the PPK antiserum was preincubated with the neuropeptide corazonin (10–4 M) exhibited PK-LI that was comparable to standard PK-LI without any preincubation, such as that shown in Fig. 1 (n = 5). Although we did not perform preadsorption controls with every neuropeptide that has been localized to this system, it is noteworthy that the PK-LI distribution in the STNS was distinct from that of previously identified neuropeptides. For example, the absence of PK-LI in the OG distinguished it from immunolabeling for proctolin, RPCH, allatostatins, orcokinins, and FLRFamides (Li et al. 2002; Marder et al. 1987; Nusbaum and Marder 1988, 1989a; Skiebe and Schneider 1994), whereas the lack of PK-LI in the ions separated it from CabTRP Ia (Blitz et al. 1995). Similarly, like corazonin, immunolabeling for the peptides CCAP and PevPK is evident in the POs but not in the STNS (Christie et al. 1995; Saideman et al. 2006).
Immunoassay screening for pyrokinin-like peptides in C. borealis tissue extract
PK peptides share the conserved C-terminal pentapeptide sequence FXPRLamide, where X is a variable amino acid (Torfs et al. 2001). The PK antiserum that we used binds with this conserved sequence. Using this antiserum, we performed immuno-dot blots on tissue extracts and HPLC fractions from these extracts. To assess the specificity of labeling in these experiments, we determined the relative ability of several PK peptides, as well as the unrelated peptide Pevkinin-2 (DFSAWAamide) (Saideman et al. 2006; Torfs et al. 1999), to generate an immunopositive result with this method. The dot blots containing Pevkinin-2 (1.4 x 10–6 M) exhibited no immunoreactivity to the PK antiserum (n = 4). In contrast, LPK (1.1 x 10–6 M; n = 3), PPK-2 (9.8 x 10–7 M; n = 17), CabPK-I (9.5 x 10–7 M; n = 1), and CabPK-II (9.7 x 10–7 M; n = 1) each showed immunostaining, which was considerably weaker with LPK than with the other three peptides.
We performed immunoaffinity-based dot blots on tissue extracts from the C. borealis brain (n = 6), thoracic ganglion (n = 1), CoG (n = 2), STG (n = 1), and PO (n = 1). We obtained positive results from every sample of tissue extract tested, with immunostaining appearing strongest for the brain extracts (data not shown). We also used this dot-blot–screening assay to identify several HPLC fractions containing PK peptide candidates for further characterization. Specifically, we screened for and identified immunopositive HPLC fractions from C. borealis extracts of brain (n = 6) and CoG tissue (n = 1). MALDI FTMS profiling was then used to reveal the peptide content of the immunoreactive fractions (brain, Fig. 2A; CoG, Fig. 2B). The immunoreactive species eluted at the same retention time for both brain and CoG extracts, suggesting that the same peptides occurred in both locations. However, because of the complexity of the tissue extracts, for both tissues multiple peptide peaks coeluted and thus were detected in individual HPLC fractions. By determining the masses in common between the two HPLC fractions, a list of candidate masses was composed for further sequencing.
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Given the complexity of the peptide profiles observed in the HPLC fractions, capillary liquid chromatography (LC) coupled with electrospray ionization (ESI) quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) was performed on the immunoreactive HPLC fractions to further separate peptide mixtures and obtain peptide sequence information (Fu and Li 2005; Fu et al. 2005). Using this approach, two novel CabPKs were de novo sequenced. These CabPK peptides had molecular masses of 1050.69 and 1036.65 Da. The MS/MS fragmentation spectrum of the first peptide (molecular mass 1050.69) from brain extract is shown in Fig. 3A. On automatic sequencing by PepSeq software, a sequence of TNFAFSPRLamide was determined. Although the b5–b8 ions were absent, a complete series of intense y-type ions enabled the assignment of the peptide sequence. In the low-mass region, immonium ions m/z 70.069, 86.09, and 120.101 indicated the presence of, respectively, proline, leucine/isoleucine, and phenylalanine.
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The MS/MS fragmentation spectrum of the second peptide (molecular mass = 1036.65 Da) is shown in Fig. 3B. We noticed that, in this spectrum, a series of fragmentation ions such as m/z 384.3, 471.3, 618.4, 689.4, and 836.5 were the same as those observed in the MS/MS spectrum of CabPK-I and in a comparable pattern. In the MS/MS spectrum of CabPK-I, these fragment ions were assigned as y3 (384.3), y4 (471.3), y5 (618.4), y6 (689.4), and y7 (836.5), respectively. This observation suggested that the C-terminal sequence of this 1036.65-Da peptide shared the identical sequence of FAFSPRLamide with CabPK-I. Although the b1 and b2 ions were weak and the y9 ion was missing, attributed to glycine in the sequence that generally produces less abundant fragment ions on cleavage (Papayannopoulos 1995), several internal fragment ions including 115.091 (GG) enabled us to assign the N-terminal sequence as SGG. The second novel peptide, with the sequence SGGFAFSPRLamide, was thus designated as CabPK-II.
Identification of CabPK-I and CabPK-II in the C. borealis STNS by MALDI FTMS
With the sequences of CabPK-I and CabPK-II determined, direct tissue MALDI FTMS was used to map the distribution of these two novel crustacean pyrokinin peptides in the C. borealis STNS. MALDI FTMS spectra revealed peaks that were consistent with the presence of both peptides in the CoG (Fig. 4A) and STG (Fig. 4B). As expected, numerous peptides were detected in both tissues. Even though there was only weak immunostaining observed in the dot blot resulting from the extraction of 100 animals’ worth of STG, both CabPK-I ([M + H]+ = 1051.570) and CabPK-II ([M + H]+ = 1037.558) were detected in the STG mass profiling by MALDI FTMS. Using internal calibration, both peptides were determined with mass measurement accuracy (MMA) of 1.59 and 5.17 ppm (n = 3), respectively. (Note: In an effort to gain sensitivity to observe low-abundance peptides, the ion cyclotron resonance cell can be overloaded with higher abundance peptides. This approach slightly degrades the MMA in complex spectra with a large concentration dynamic range.) Similarly, CabPKs were observed in the CoG with MMA of 0.124 ppm for CabPK-I and 2.30 ppm for CabPK-II. Finally, we also identified CabPK-II in the LC fraction of C. borealis PO extract. SORI-CID (sustained off-resonance irradiation collision-induced) tandem mass spectrometric analysis was used to confirm the CabPK-II amino acid sequence from the HPLC fraction of the PO extract (data not shown). CabPK-I was not detected in the POs, suggesting that only CabPK-II was expressed in the POs. However, the lack of CabPK-I detection instead might have resulted from analyte suppression and/or a lower level of this isoform in the PO tissue.
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In most isolated STGs, the influence of PK (10–6 M) superfusion on the pyloric rhythm was limited to an excitation of the inferior cardiac (IC) and ventricular dilator (VD) neurons (Fig. 5). These are the two pyloric neurons that also participate in the gastric mill rhythm (Blitz and Nusbaum 1997; Weimann et al. 1991). As is evident from Fig. 6 and subsequent results (see following text), all tested PKs, including CabPK-I, CabPK-II, PPK, and LPK, had comparable actions when each was superfused at 10–6 M. For example, none of these PKs elicited a consistent change in pyloric cycle frequency (Saline: 0.62 ± 0.22 Hz; PPK/LPK: 0.66 ± 0.25 Hz; CabPKI/II: 0.52 ± 0.13 Hz; n = 10–18, ANOVA, P > 0.05).
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; Nusbaum and Marder 1989b), we also determined whether the combined results reported above were skewed by the fact that relatively fast control rhythms were affected less than weakly cycling rhythms. We therefore plotted the pyloric cycle frequency during PK application as a function of its cycle frequency immediately before PK application for each preparation. Doing so revealed that none of the PKs altered the pyloric cycle frequency, regardless of the control frequency (Fig. 6A). Similarly, PK application did not alter the mean number of lateral pyloric (LP) neuron spikes per burst across preparations (Saline: 3.6 ± 2.1; PPK/LPK: 5.1 ± 2.9; CabPKI/II: 2.8 ± 1.3; n = 10–15, ANOVA, P > 0.05), regardless of whether the preapplication level of LP activity was relatively weak or strong (Fig. 6B). In contrast to the lack of PK influence on pyloric cycle frequency and LP neuron activity, PK application consistently increased IC and VD neuron activity. Specifically, the number of spikes/burst in the IC neuron increased in the presence of all applied PKs (Saline: 1.2 ± 1.5; PPK/LPK: 4.3 ± 1.7; CabPKI/II: 5.5 ± 2.4; n = 10–14, ANOVA, P < 0.05). The same was the case for the VD neuron (Saline: 0.05 ± 0.2; PPK/LPK: 4.7 ± 1.9; CabPKI/II: 3.7 ± 2.2; n = 9–14, ANOVA, P < 0.05). In the isolated STG, the IC neuron was either silent or weakly active during saline superfusion, whereas the VD neuron was generally silent (e.g., Fig. 5). PK excitation of IC appeared to be equally effective whether it was silent or active before PK application (Fig. 6C). Similarly, PK superfusion consistently activated the VD neuron (Fig. 6D).
Pyrokinins excite gastric mill neurons
Superfusing any of the examined PKs in normal C. borealis saline routinely excited many gastric mill neurons, including the gastric mill protractor phase neurons LG (n = 252), IC (n = 205; Figs. 5 and 6), MG (n = 12) and the retractor phase neurons Int1 (n = 271), VD (n = 218; Figs. 5 and 6), and DG (n = 264) (Saideman 2006). This PK-mediated excitation usually coincided with its activation of the gastric mill rhythm (see following text). The two remaining types of gastric mill neurons, including the gastric mill (GM: n = 17) protractor neuron and the anterior median (AM: n = 5) retractor neuron, were never activated by PK peptide application when they were at their normal resting potential (about –60 mV), which is subthreshold for action potential generation. However, these two neurons each exhibited increased activity during PK superfusion if they were sufficiently depolarized to be suprathreshold when PK was applied (GM: n = 17; AM: n = 6) (data not shown). Thus the activity of all gastric mill neurons was enhanced by PK peptides superfused in normal saline.
In addition to exciting the gastric mill neurons, PK superfusion also upregulated the intrinsic properties of some of these neurons. For example, the PKs unmasked the ability of the DG neuron (n = 11) to generate a plateau potential (Fig. 7A). During PK superfusion, a brief depolarizing current injection into DG evoked persisting spike activity that outlasted the current pulse (Fig. 7A). As is typical for plateau potentials (Kiehn 1991; Russell and Hartline 1978), injection of a brief but sufficiently strong hyperpolarizing current pulse prematurely suppressed these plateau responses in the DG neuron. The duration of DG plateaus tended to be longer in than that of its gastric mill–timed bursts. PK superfusion also elicited plateau potentials in the LG neuron (data not shown; see Saideman 2006).
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; Marder and Paupardin-Tritsch 1978). In fact, the nonglutamatergic STG neurons (the cholinergic VD, LPG, and two PD neurons) are largely follower motor neurons in C. borealis with modest (PD) or no (VD, LPG) evident transmitter-mediated synaptic actions within the STG, during superfusion with either normal saline or PTX saline (Saideman and Nusbaum, unpublished observations). Thus when PTX was applied to the isolated STG, there were few active transmitter-mediated synapses remaining between the STG neurons, although electrical coupling persisted. Consequently, during PTX superfusion only the electrically coupled pyloric pacemaker ensemble (AB, PD, LPG) retained their pyloric-timed activity. For example, the gastric mill neurons that normally exhibited pyloric-timed activity (e.g., Int1, IC, VD) during normal saline superfusion lost that activity pattern during PTX superfusion and instead exhibited either tonic or intermittent activity (Fig. 8A). This was also the case for the MG neuron (n = 4; not shown). Additionally, for some neurons such as IC, their level of spontaneous activity increased in PTX (Fig. 8A). As is evident in Fig. 8, during PTX superfusion the gastric mill neuron activity patterns were largely unrelated to one another. Many gastric mill neurons remained responsive to PK (10–6 M) application during PTX superfusion, albeit with altered activity patterns relative to those in normal saline (Fig. 8). The VD neuron firing frequency was increased by PK application in PTX saline (PTX: 1.3 ± 2.4 Hz; PK/PTX: 3.4 ± 2.5 Hz, n = 4, P < 0.05). Under this condition, Int1 also responded, reversibly, to PK application with a membrane potential depolarization and increased firing rate (PTX: 6.8 ± 3.1 Hz; PK/PTX: 9.9 ± 2.4, n = 3, P < 0.05). For example, in Fig. 8A, the Int1 firing frequency increased from 6.7 ± 0.7 Hz in PTX alone to 9.5 ± 0.4 Hz when PK and PTX were coapplied. The Int1 firing frequency then returned to 4.0 ± 1.2 Hz when the preparation was returned to PTX alone. In contrast, IC neuron activity was not enhanced by PK application in PTX saline (PTX: 7.1 ± 2.3 Hz, PK/PTX: 6.3 ± 0.7 Hz, n = 5, P > 0.05).
Each of the other gastric mill neurons also remained responsive to PK (10–6 M) application during PTX superfusion, albeit with neuron type-specific responses. For example, whereas the protractor LG neuron tended to be subthreshold during PTX superfusion, the addition of a PK peptide consistently, and reversibly, elicited gastric mill rhythm-like bursting from this neuron (n = 11) (Fig. 8, A and B). This result was surprising because there was no indication of PK-elicited intrinsic oscillatory activity in the LG neuron in normal saline (Saideman 2006). This rhythmic bursting of the LG neuron could not have been the result of persisting rhythmic synaptic inhibition from the retractor phase neuron Int1 because, although LG and Int1 are reciprocally inhibitory and normally burst in alternation during the gastric mill rhythm, both neurons make PTX-sensitive synapses and therefore fired independently during PTX superfusion (Fig. 8A). It is also noteworthy that, despite the presence of electrical coupling between the protractor phase neurons (LG, GM, MG, IC), only the LG neuron responded to PK application in PTX with a rhythmic bursting pattern. At these times, the IC (Fig. 8A) and MG neurons fired tonically, whereas the GM neurons remained silent (not shown). Rhythmic bursting was also consistently, and reversibly, elicited in the DG neuron by PK application in the presence of PTX (n = 12) (Fig. 8B). Note that at these times the DG and LG neuron bursts were independent of each other (Fig. 8B). The cycle period of the PK-elicited rhythmic bursting in the DG neuron was comparable during normal saline and PTX superfusion (PK/Normal Saline: 47.2 ± 8.6 s; PK/PTX: 31.8 ± 16.6 s; n = 4, P > 0.05).
Last, as was also the case during normal saline superfusion, the gastric mill (GM) and anterior median (AM) neurons were silent during PTX superfusion. Under these conditions PK application elicited no response from either neuron (GM: n = 15; AM: n = 3). We did not test whether these two neurons exhibited the same voltage-dependent excitatory response to PK application in PTX that occurred when this peptide was applied in normal saline. Thus PK application increased the activity in at least five of the eight types of gastric mill neurons, even when most intracircuit synapses were suppressed.
Pyrokinins activate the gastric mill rhythm
PK peptide superfusion in normal saline effectively activated rhythmic neuronal activity from the gastric mill circuit. Specifically, superfusion of either CabPK-I (n = 14), CabPK-II (n = 13), PPK (n = 31), or LPK (n = 136) (10–6 M) to the isolated STG routinely and reversibly activated alternating bursting from the gastric mill CPG neurons LG and Int1 (Fig. 9). The cycle period for this rhythmic activity (16.8 ± 9.2 s, n = 50) was similar to that for gastric mill rhythms elicited by other pathways (Bartos et al. 1999; Beenhakker and Nusbaum 2004; Wood et al. 2004). This gastric mill rhythm activity was consistently accompanied by appropriately coordinated bursting of the MG and IC protractor phase neurons and VD retractor phase neuron (n = 191) (Figs. 9 and 10). Neither the GM nor AM neurons participated in this PK-elicited rhythm (n = 194). The retractor neuron DG was also consistently activated to generate rhythmic action potential bursts (n = 168). However, the rhythmic DG bursts were often not time-locked with those of the other gastric mill neurons (n = 157/168) (Fig. 9; see also Fig. 13). Although in Fig. 9 there was a regular pattern in which there was a single DG burst for every two LG bursts, this was a rare occurrence (n = 4/168). In some preparations (n = 11/168), however, PK (10–6 M) application elicited a fully coordinated gastric mill rhythm. At these times, the DG neuron bursts were appropriately time-locked with those of the other gastric mill neurons in every gastric mill cycle (Fig. 11). The cycle period exhibited by these fully coordinated rhythms (16.2 ± 4.5 s; n = 9) was comparable to the cycle period occurring when the DG bursts were not coordinated with the other gastric mill neurons (P > 0.05, ANOVA).
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; Coleman et al. 1995). In contrast, the DG bursting was generally not time-locked with that of other gastric mill neurons on a cycle-by-cycle basis and appeared to burst independently. As shown in Fig. 12, the effective threshold concentration for these CabPK actions was >10–8 M. Moreover, the cycle period of both the LG and DG bursting was the same at 10–7 and 10–6 M CabPK (Fig. 12). The same was true for the number of spikes per burst in the LG neuron but not in the DG neuron, where the number of spikes per burst was significantly higher at 10–6 M CabPK (Fig. 12). Pyrokinin excites the MCN1 axon proximal to the STG
In some experiments (n = 22/194), PK superfusion (10–6 M) activated the projection neuron MCN1 (Fig. 13). In the subset of experiments where we simultaneously monitored the activity of each MCN1 (n = 13/22), PK superfusion activated both of them in only three preparations. MCN1 was never activated by lower PK concentrations (n = 9). This activation of MCN1 occurred despite the fact that the CoGs had been removed, thereby eliminating the MCN1 somata, their CoG arborizations, and their normal spike initiation zones. Despite the absence of the CoGs, each MCN1 was readily identified in extracellular recordings (ion; Fig. 13) (Bartos and Nusbaum 1997; Coleman and Nusbaum 1994). In some experiments, MCN1 activity was further identified by the presence of unitary electrical excitatory postsynaptic potentials (eEPSPs) in the LG neuron that were time-locked with the action potentials recorded in the ion (see following text) (Coleman et al. 1995).
In preparations where PK peptide activated MCN1, the latency to the start of its activity always lagged considerably relative to PK excitation of the gastric mill neurons. In general, the gastric mill neurons were excited within a few minutes (3.6 ± 0.9 min, n = 22) after the start of PK superfusion, with a considerable fraction of this duration resulting from the time for the peptide to clear the superfusion line, enter the bath, and reach an effective bath concentration. In contrast, PK activation of MCN1 exhibited a latency of 8.1 ± 4.6 min (n = 22). Moreover, whereas PK excitation of the gastric mill neurons persisted well into the post-PK washout, PK activation of MCN1 was transient and usually terminated before the end of PK superfusion.
When activated by PK superfusion, MCN1 fired at a relatively low frequency (range: 3–15 Hz). Nonetheless, this firing frequency was sufficient to influence the gastric mill rhythm (Fig. 13). In preparations where PK application resulted in DG neuron bursting separately from the other gastric mill neurons, there were two different outcomes of subsequent PK activation of MCN1. In some experiments (n = 15/22), the DG neuron continued to burst independently but its activity, as well as that of the other rhythmically active gastric mill neurons, was enhanced (Fig. 13A). In the other seven preparations, subsequent activation of MCN1 switched the gastric mill pattern to one where the DG bursts became time-locked to the other gastric mill neurons (n = 7/22) (Fig. 13B). This distinction did not correlate with the MCN1 firing frequency elicited by PK.
Interestingly, PK activation of MCN1 often occurred in one of the nerves anterior to the STG. As indicated above, this activation did not occur at the usual site of MCN1 excitation because the CoGs had been removed. Three lines of evidence supported the hypothesis that pyrokinin activation of MCN1 did not occur in the STG. First, when MCN1 action potentials are initiated in the STG and propagate antidromically, they are rhythmically suppressed during each gastric mill protraction phase (Coleman and Nusbaum 1994; Nusbaum et al. 1992). This is explained by the fact that the LG neuron presynaptically inhibits the MCN1 terminals in the STG (Coleman and Nusbaum 1994). In contrast, when MCN1 action potentials initiate in the CoG, they propagate past the ion recording site before they enter the STG and are affected by the inhibitory synapse from LG (Coleman and Nusbaum 1994). In most of our experiments where PK activated MCN1 along with rhythmic LG neuron bursting, MCN1 activity in the ion was tonic instead of being suppressed during each LG burst (n = 11/15) (Fig. 13, A and B). In only a few preparations (n = 4/15) was MCN1 activity rhythmically suppressed during each LG burst, indicating that in these instances the MCN1 action potentials had been initiated within the STG (Fig. 13C).
A second indication that PK peptide application activated MCN1 anterior to the STG came from preparations in which MCN1 activity in the ion was tonic during rhythmic LG bursting. Specifically, at these times there was a measurable delay (26 ± 3 ms, n = 4) from each PK-activated MCN1 action potential in the ion to the onset of the resulting eEPSP in the LG neuron (Fig. 13D). This latency was comparable to that previously measured when MCN1 was stimulated extracellularly in the ion (Coleman et al. 1995). In contrast, in preparations where the evidence supported PK activation of MCN1 spikes in the STG, each eEPSP in the LG neuron remained time-locked to a MCN1 spike in the ion but preceded that spike by 24 ± 5 ms (n = 4) (Fig. 13D). Last, in one experiment during PK activation of MCN1, we transected the ion on the stn side of the extracellular ion recording site. Because this ion had been transected between the ion recording site and the CoG at the start of the experiment, this second transection completely separated this region of ion from the rest of the STNS. Nonetheless, the MCN1 axon activity in this isolated ion was reversibly activated to fire tonically by PK superfusion.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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The combined MS results and PK-LI support the presence of CabPKs in the neuropil of both the STG and CoGs and in a subset of CoG neuronal somata. The lack of PK-LI in any STG somata suggested that the CabPK present in the STG occurs in the axon terminals of two or three bilaterally symmetric CabPK-containing CoG projection neurons. The occurrence of CabPK in the POs suggests that this peptide gains access to the STG circuits as a circulating hormone as well as by local release from modulatory projection neurons. There are a number of previously identified neuroactive peptides that share these access routes to the STG with the CabPKs (Marder and Bucher 2006; Marder et al. 2005).
The immuno-dot-blot assay enabled us to screen HPLC fractions of complex tissue extracts, providing a more targeted discovery of endogenous CabPKs. The combination of immunoreactivity profiling with MALDI FTMS screening produced a short list of putative PK peptide candidates for further sequencing analysis. Despite the high chemical complexity of the samples, nanoscale LC ESI-QTOF MS/MS allowed us to fragment each individual peptide while eluting from nanoLC columns, leading to the identification of each CabPK.
Pyrokinin peptide family members share an extended carboxy-terminal sequence of FXPRLamide, with X representing a variable amino acid (Altstein 2004). The CabPKs are particularly closely related to PPK 1 (DFAFSPRLamide), sharing sequence identity for the seven carboxy-terminal amino acids (Torfs et al. 2001). The carboxy terminal appears to be the biologically active region of the PK peptides (Altstein 2004). This hypothesis is supported by our finding
