Identification and characterization of a tachykinin-containing neuroendocrine organ in the commissural ganglion of the crab Cancer productus
1 Department of Biology, University of Washington, Box 351800, Seattle, WA
98195-1800, USA
2 Friday Harbor Laboratories, University of Washington, 620 University Road,
Friday Harbor, WA 98250, USA
3 Department of Chemistry, University of WisconsinMadison, 1101
University Avenue, Madison, WI 53706-1369, USA
4 Department of Physics, Santa Clara University, 500 El Camino Real, Santa
Clara, CA 95053-0315, USA
5 School of Pharmacy, University of WisconsinMadison, 777 Highland
Avenue, Madison, WI 53705-2222 USA
* Author for correspondence (e-mail: crabman{at}u.washington.edu)
Accepted 7 July 2005
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: stomatogastric nervous system, hormone, APSGFLGMRamide, Cancer borealis tachykinin-related peptide Ia, CabTRP Ia, anterior commissural organ, ACO, laser-scanning confocal microscopy, mass spectrometry, MALDI-FTMS
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In most invertebrates, particularly insects, large numbers of
species-specific TRP isoforms are common
(Nachman et al., 1999;
Nässel, 1999
;
Vanden Broeck et al., 1999
;
Severini et al., 2002
). For
example, 10 TRPs have been isolated from the cockroach Leucophea
maderae (Muren and Nässel,
1996
,
1997
), seven from the honeybee
Apis mellifera (Takeuchi et al.,
2003
), five from the locust Locusta migratoria (Schoofs
et al.,
1990a
,b
,
1993
), five from the fruit fly
Drosophila melanogaster (Siviter
et al., 2000
) and three from the mosquito Culex
salinarius (Meola et al.,
1998
). In contrast to the diversity of species-specific TRPs
present in insects, only a single isoform is thought to be present in decapod
crustaceans (Christie et al.,
1997a
; Nieto et al.,
1998
; Li et al.,
2002a
; Huybrechts et al.,
2003
; Yasuda-Kamatani and
Yasuda, 2004
). This peptide, APSGFLGMRamide or Cancer
borealis tachykinin-related peptide Ia (CabTRP Ia), has been isolated
from or is predicted by cDNA to be present in the crab Cancer
borealis, the shrimp Panaeus vannamei, the chelate marine
lobster Homarus americanus, the freshwater crayfish Procambarus
clarkii and the spiny lobster Panulirus interruptus
(Christie et al., 1997a
;
Nieto et al., 1998
;
Li et al., 2002a
;
Huybrechts et al., 2003
;
Yasuda-Kamatani and Yasuda,
2004
).
While no antibody has been generated directly against CabTRP Ia, the
peptide has been shown to cross-react with a rat monoclonal antibody to
substance P and with several antibodies generated against insect TRPs
(Cuello et al., 1979;
Nässel, 1993
;
Christie et al., 1997a
;
Winther and Nässel,
2001
). Immunohistochemical studies using these antibodies have
shown that CabTRP Ia is widely distributed within the nervous system of
decapod crustaceans (Mancillas et al.,
1981
; Fingerman et al.,
1985
; Goldberg et al.,
1988
; Sandeman et al.,
1990a
,b
;
Schmidt and Ache, 1994
,
1997
; Blitz et al.,
1995
,
1999
; Christie et al.,
1995a
,
1997a
,b
;
Schmidt,
1997a
,b
;
Langworthy et al., 1997
;
Fénelon et al., 1999
;
Glantz et al., 2000
;
Thirumalai and Marder, 2002
;
Pulver and Marder, 2002
). In
C. borealis, P. interruptus and H. americanus, one area of
the nervous system that exhibits TRP immunoreactivity is the stomatogastric
nervous system (STNS; Fig. 1),
which controls the movement of the foregut musculature
(Goldberg et al., 1988
; Blitz
et al., 1995
,
1999
; Christie et al.,
1997a
,b
;
Fénelon et al.,
1999
).
|
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
For tissue collection, crabs were anesthetized by packing in ice for 3060 min, after which the dorsal carapace was removed and the foregut isolated from the animal. Following extraction of the foregut, the CoGs, and in some cases the entire STNS, were dissected free in chilled (approximately 10°C) physiological saline [composition in mmol l1: 440 NaCl; 11 KCl; 13 CaCl2; 26 MgCl2; 10 Hepes acid, pH 7.4 (adjusted with NaOH)]. For some crabs, the eyestalks and dorsolateral walls of the pericardial chamber were also removed. From these structures, we isolated two well-known neuroendocrine organs: the sinus glands (SGs) and pericardial organs (POs).
Whole-mount immunohistochemistry
Immunohistochemistry was performed as whole mounts. Specifically, dissected
tissue was pinned in a Sylgard 184 (World Precision Instruments, Inc.,
Sarasota, FL, USA; catalog #SYLG184)-lined Petri dish and fixed in a solution
of either 4% paraformaldehyde (EM grade; Electron Microscopy Sciences,
Hatfield, PA, USA; catalog #15710) in 0.1 mol l1 sodium
phosphate (P) buffer (pH 7.4), 4%
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC; Sigma-Aldrich, St Louis,
MO, USA; catalog #E-7750) in P, 4% paraformaldehyde and 1% EDAC in P or 100%
methanol (HPLC grade; Sigma-Aldrich; catalog #27047-4), depending on the
primary antibody being used (see below). All solutions containing
paraformaldehyde or EDAC were prepared immediately prior to use, and tissue
was fixed at 4°C. Methanol fixation was done at 20°C.
Regardless of solution, tissues were fixed for 1224 h, except as noted
below (see Primary antibodies). Following fixation, tissue was rinsed five
times over approximately five hours in a solution of P containing 0.3% Triton
X-100 (P-Triton). Incubation in primary antibody (see below) was carried out
in P-Triton, with 10% normal donkey serum (NDS; Jackson ImmunoResearch
Laboratories, Inc., West Grove, PA, USA; catalog #017-000-121) added to
diminish nonspecific binding. Following incubation in primary antibody, tissue
was again rinsed five times over approximately five hours in P-Triton and then
incubated in a 1:300 dilution of secondary antibody (see below) for
1224 h. As with the primary antibody, incubation with secondary
antibody was performed in P-Triton containing 10% NDS. After incubation in
secondary antibody, preparations were rinsed five times over approximately
five hours in P and mounted between a glass microscope slide and cover slip
using Vectashield mounting medium (Vector Laboratories, Inc., Burlingame, CA,
USA; catalog #H-1000). Incubations in primary and secondary antibodies were
done using gentle agitation at 4°C. All tissues were rinsed at room
temperature (1824°C) without agitation. Secondary antibody
incubation was conducted in the dark, as was all subsequent processing.
Likewise, slides were stored in the dark at 4°C until examined.
Primary antibodies
Each of the primary antibodies employed in our study has been used
previously to map the distribution of its respective antigen in
crustacean/insect nervous systems and has been shown to be specific for its
antigen. The references provided for each antibody describe its
development/specificity and/or use in arthropod neural tissue.
For the detection of TRP, a rat monoclonal antibody to substance P (clone
NC1/34 HL; Abcam Incorporated, Cambridge, MA, USA; catalog # ab6338;
Cuello et al., 1979;
Goldberg et al., 1988
;
Christie et al., 1997a
) was
used at a final dilution of 1:300. For the detection of the small molecule
transmitter
-aminobutryic acid (GABA), a rabbit polyclonal antibody
(Sigma-Aldrich; catalog #A2052; Blitz et
al., 1999
; Swensen et al.,
2000
) was used at a final dilution of 1:500. For the detection of
the amine dopamine, a mouse monoclonal antibody to its biosynthetic enzyme
tyrosine hydroxylase (Immunostar Inc., Hudson, WI, USA; catalog # 22941;
Tierney et al., 1999
;
Pulver and Marder, 2002
) was
used at a final dilution of 1:1000. For the detection of the amine histamine,
a rabbit polyclonal antibody (Immunostar; catalog # 22939;
Panula et al., 1988
;
Christie et al., 2004b
) was
used at a final dilution of 1:500. For the detection of the amine serotonin, a
rabbit polyclonal antibody (Immunostar; catalog # 20080;
Tierney et al., 1999
;
Pulver and Marder, 2002
) was
used at a final dilution of 1:500. To assay for the gas carbon monoxide, a
rabbit polyclonal antibody to its biosynthetic enzyme heme oxygenase 2
(Stressgen Biotechnologies Corp, Victoria, BC, Canada; catalog # OSA-200;
Christie et al., 2003
) was
used at a final dilution of 1:100. To assay for the gas nitric oxide, a rabbit
polyclonal antibody to its biosynthetic enzyme nitric oxide synthase (Affinity
Bioreagents, Golden, CO, USA; catalog # PA1-039;
Christie et al., 2003
) was
used at a final dilution of 1:300. For the detection of allatostatin-like
peptides, a mouse monoclonal antibody (Dr B. Stay, University of Iowa, Iowa
City, IA, USA; Stay et al.,
1992
; Woodhead et al.,
1992
; Pulver and Marder,
2002
) was used at a final dilution of 1:100. For the detection of
buccalin-like peptides, a rabbit polyclonal antibody (Dr K. Weiss, Mount Sinai
School of Medicine, New York, NY, USA;
Miller et al., 1992
;
Christie et al., 1994
) was
used at a final dilution of 1:100. For the detection of cholecystokinin
(CCK)-related peptides, a rabbit polyclonal antibody (Dr G. Turrigiano,
Brandeis University, Waltham, MA, USA;
Turrigiano and Selverston,
1991
; Christie et al.,
1995b
) was used at a final dilution of 1:300. For the detection of
corazonin-related peptides, a rabbit polyclonal antibody (Dr J. Veenstra,
Université Bordeaux 1, Talence cedex, France;
Veenstra, 1991
;
Christie and Nusbaum, 1995
)
was used at a final dilution of 1:500. For the detection of crustacean
cardioactive peptide (CCAP), a rabbit polyclonal antibody (Dr H. Dircksen,
Stockholm University, Stockholm, Sweden;
Dircksen and Keller, 1988
;
Stangier et al., 1988
;
Christie et al., 1995a
) was
used at a final dilution of 1:500. For the detection of FLRFamide-related
peptides, a rabbit polyclonal antibody (Immunostar; catalog # 20091;
Christie et al., 2004a
) was
used at a final dilution of 1:300. For the detection of myomodulin-like
peptides, a rabbit polyclonal antibody (Dr K. Weiss;
Miller et al., 1991
;
Christie et al., 1994
) was
used at a final dilution of 1:300. For the detection of orcokinins, a rabbit
polyclonal antibody (Dr H. Dircksen;
Bungart et al., 1994
;
Li et al., 2002b
;
Skiebe et al., 2002
) was used
at a final dilution of 1:5000. For the detection of ß-pigment dispersing
hormone (ß-PDH)-related peptides, a rabbit polyclonal antibody (Dr K.
Rao, University of West Florida, Pensacola, FL, USA;
Dircksen et al., 1987
;
Mortin and Marder, 1991
) was
used at a final dilution of 1:1000. For the detection of proctolin, a rabbit
polyclonal antibody (Dr D. Nässel, Stockholm University; code K9832/13;
Johnson et al., 2003
) was used
at a final dilution of 1:500. For the detection of red pigment concentrating
hormone (RPCH), a rabbit polyclonal antibody (Dr R. Elde, University of
Minnesota, Minneapolis, MN, USA; Madsen et
al., 1985
; Nusbaum and Marder,
1988
) was used at a final dilution of 1:300.
The fixative used for all antibodies except for those generated against tyrosine hydroxylase, histamine and CCAP was 4% paraformaldehyde (tissue for GABA immunoprocessing fixed for 23 h only). Fixation for tyrosine hydroxylase utilized 100% methanol, while 4% EDAC was employed for histamine, and a combination of 4% paraformaldehyde and 1% EDAC was used for CCAP.
Secondary antibodies
The secondary antibodies used in our experiments were donkey anti-rat
immunoglobulin G (IgG) conjugated to Alexa Fluor 488 (Molecular Probes,
Eugene, OR, USA; catalog #A-21208) or Alexa Fluor 594 (Molecular Probes;
catalog #A-21209), donkey anti-rabbit IgG conjugated to Alexa Fluor 488
(Molecular Probes; catalog #A-21206) or Alexa Fluor 594 (Molecular Probes;
catalog #A-21207) and donkey anti-mouse IgG conjugated to Alexa Fluor 488
(Molecular Probes; catalog #A-21202) or Alexa Fluor 594 (Molecular Probes;
catalog #A-21203).
Confocal and epifluorescence microscopy
Fluorescently labeled tissue was viewed and data collected using one of two
Bio-Rad MRC 600 laser scanning confocal microscopes (Bio-Rad Microscience
Limited, Hemel Hempstead, UK), a Bio-Rad Radiance 2000 laser scanning confocal
microscope or a Nikon Eclipse E600 epifluorescence microscope (Tokyo, Japan).
The Bio-Rad MRC 600 system at Friday Harbor Laboratories is equipped with a
Nikon Diaphot inverted microscope and a krypton/argon mixed gas laser. Nikon
Fluor 10x 0.5NA dry, PlanApo 20x 0.75NA dry, Nikon Fluor 40x
0.85NA dry and PlanApo 60x 1.4NA oil immersion objective lenses and
Bio-Rad-supplied BHS, YHS and K1/K2 filter sets and Comos software were used
for imaging preparations on this system (filter specifications are as
described in Christie et al.,
1997b). The Bio-Rad MRC 600 system located at the University of
Washington (Department of Biology) is equipped with a Nikon Optiphot upright
microscope and, with the exception of the Nikon Fluor 40x 0.85NA dry
lens, uses the same laser, filters, software and objective lenses as the MRC
600 system located at Friday Harbor Laboratories. The Bio-Rad Radiance 2000
system is equipped with a modified Nikon Eclipse E600FN microscope and a
krypton/argon mixed gas laser (568 nm excitation line used). For imaging on
this system, Nikon PlanApo 10x 0.45NA DIC dry, PlanApo 20x 0.75NA
DIC dry and PlanApo 60x 1.4NA DIC oil immersion objective lenses as well
as a Bio-Rad-supplied E600LP emission filter and Bio-Rad LaserSharp 2000
software were used. The Nikon Eclipse E600 epifluorescence microscope is
equipped with Nikon PlanFluor 10x 0.30NA, PlanFluor 20x 0.50NA and
PlanFluor 40x 0.75NA dry objective lenses and ENDOW GFP HYQ (EX,
450490 nm; DM, 495 nm; BA, 500550 nm) and G-2E/C TRITC (EX,
528553 nm; DM, 565 nm; BA, 600660 nm) filter sets.
India ink mapping of the circulatory system
India ink injection into the circulatory system has long been used to map
the distribution of blood vessels in neural tissue
(Lane et al., 1981;
Renkin et al., 1981
;
Renkin, 1985
;
Farley, 1990
;
Hogers et al., 1995
;
Murray and Wilson, 1997
;
Grivas et al., 2003
;
Sasaki et al., 2003
;
Cerri et al., 2004
;
Hutchinson and Savitzky, 2004
;
Marinkovic et al., 2004
).
Here, we developed a method using this reagent to visualize the distribution
of hemolymph vessels and lacunae in the stomatogastric neuromuscular system.
Specifically, Higgins Fountain Pen India ink (Eberhard Faber Inc., Lewisberg,
TN, USA; catalog #723) or Koh-I-Noor Fount India drawing ink (Koh-I-Noor,
Bloomsbury, NJ, USA; catalog #9150-D) was dried down and then reconstituted in
a similar amount of physiological saline. The resulting saline/ink solution
was injected into the pericardial chamber using a 22-gauge needle attached to
a 1-ml plastic syringe via penetration through the junction of the
thorax and abdomen. For small animals (<120 g), 200 µl of saline/ink
solution was injected into each individual. Intermediate-sized animals
(120250 g) were injected with 500 µl and large animals (>250 g)
received a 1 ml injection of the saline/ink solution. Following the injection,
animals were returned to their tanks for 14 h, then anesthetized and
dissected as described earlier. Nervous system tissue was then pinned flat in
a Sylgard-lined Petri dish containing physiological saline, and ink
infiltration into lacunae was visualized using either a Nikon SMZ800 or Nikon
SMZ1000 stereomicroscope with incident illumination provided by a Fiber-Lite
Model 190 fiber optic illuminator (Dolan-Jenner Industries, Inc., Woburn, MA,
USA). Micrographs of ink infiltration were taken using either a Nikon CoolPix
4500 digital camera mounted on the SMZ800 microscope or a Nikon CoolSNAP
digital camera mounted on the SMZ1000 microscope.
Coincidence of ink filling and tachykinin-like immunoreactivity
Following ink infiltration, some CoGs were fixed and immunoprocessed with
the substance P antibody to assess the coincidence of the hemolymph lacunae
and the TRP immunoreactivity. These ink-filled/substance P immunoprocessed
ganglia were imaged using the Bio-Rad Radiance 2000 confocal system, with
ink-filling visualized via its transmitted light detector.
Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry
Anterior medial quadrant of the CoG
For the mass spectrometric identification of TRP isoforms in the
tachykininergic plexus of the CoG, ganglia were dissected and pinned in a
Sylgard-lined Petri dish containing chilled physiological saline. Incident
illumination was used to identify the location of the plexus in each ganglion,
then the site was subsequently isolated and placed in acidified methanol [90%
methanol (HPLC grade; Sigma-Aldrich), 9% glacial acetic acid (sequencing
grade; Fisher Scientific, Fair Lawn, NJ, USA; catalog #BP1185-500) and 1%
water (HPLC grade; Sigma-Aldrich; catalog #27073-3)]. Tissue from 20 ganglia
was pooled in approximately 200 µl of acidified methanol and stored at
80°C until utilized for analysis.
Direct tissue analysis of individual tissue pieces was performed as
previously described (Kutz et al.,
2004). Briefly, a tissue fragment was desalted in 10 mg
ml1 2,5-dihydroxybenzoic acid (DHB; ICN Biomedicals
Incorporated, Costa Mesa, CA, USA; catalog #190209). Next, 0.2 µl of
saturated DHB solution (in 50:50 methanol:water, v/v) was added to one facet
of a matrix-assisted laser desorption/ionization (MALDI) target plate, and the
desalted tissue fragment quickly placed into the matrix. The tissue-containing
matrix was then crystallized at room temperature.
Mass spectrometric analysis of peptides was carried out using an IonSpec
HighRES MALDI Fourier transform mass spectrometer (FTMS; IonSpec Corporation,
Lake Forest, CA, USA). In order to increase the signal-to-noise ratio, in-cell
accumulation was performed to allow multiple packets of ions resulting from
the tissue sample to accumulate in the analyzer cell prior to detection. For
the detection of low-mass peptides (i.e. approximately 1000
m/z), a pulse sequence was used to more efficiently
transport ions into the analyzer cell
(Kutz et al., 2004).
Hemolymph
To determine if CabTRP Ia is a circulating hormone, we used MALDI-FTMS to
assay the hemolymph. Hemolymph was collected by inserting a 22-gauge needle
attached to a 3-ml plastic syringe through the junction of the thorax and
abdomen into the pericardial chamber. A fresh needle and syringe were used for
removal of hemolymph from each animal. Approximately 2 ml of hemolymph was
withdrawn from each animal and immediately placed in twice its volume of
acidified methanol and vortexed for 3 min at 10°C using a Thermolyne Maxi
Mix II tabletop vortexer (Barnstead/Thermolyne, Dubuque, IA, USA). Following
vortexing, the hemolymph/acidified methanol mixture was centrifuged for 5 min
at 15 800 g using an Eppendorf 5415C tabletop centrifuge
(Eppendorf AG, Hamburg, Germany), also at 10°C. After centrifugation, the
resulting supernatant was removed and stored at 80°C until utilized
for analysis.
Prior to mass spectrometric analysis, large proteins and salts were removed from the extracted hemolymph. Large proteins were removed by placing 500 µl of crude extract in a 10 000 Da molecular mass cutoff tube (Argos Technologies, catalog #VS0101) and centrifuging it at 16 100 g for 10 min at room temperature. The resulting low-molecular-mass filtrate was concentrated using a Savant SC 110 SpeedVac concentrator (Thermo Electron Corporation, West Palm Beach, FL, USA) and then resuspended in 10 µl of 0.1% formic acid (puriss grade; Sigma-Aldrich; catalog #94318). The acidified sample was desalted by passing it through a ZipTipC18 pipette tip (Millipore, Billerica, MA, USA; catalog #ZTC18S096) and eluting the bound peptides with 4 µl of 50% acetonitrile. Desalted extract was mixed 1:1 with DHB matrix on a MALDI plate and allowed to crystallize at room temperature, after which MALDI-FTMS analysis was performed as per the CoG fragments.
Muscle physiology
Neuromuscular preparations were dissected from the C. productus
foregut and pinned flat in 5-ml Sylgard-lined Petri dishes. Nerves and muscles
were identified according to the nomenclature of Maynard and Dando
(1974). During recording
sessions, the bath volume was maintained at approximately 3 ml and
preparations were continuously superfused (45 ml
min1) with physiological saline. (It should be noted that
the saline used in the physiological experiments was buffered using 11.2 mmol
l1 Trizma base and 5.1 mmol l1 maleic acid
rather than the 10 mmol l1 Hepes acid used in the saline
employed for our anatomical and mass spectrometric experiments.) CabTRP Ia was
bath-applied by means of a switching port at the inflow of the superfusion
system. This peptide was synthesized and purified using standard techniques
(Christie et al., 1997a
) by
the Cancer Research Center of the University of Pennsylvania School of
Medicine (Philadelphia, PA, USA) and was a gift from Dr Michael P. Nusbaum
(Department of Neuroscience, University of Pennsylvania School of Medicine).
Synthetic CabTRP Ia was dissolved in distilled water at a concentration of
103 mol l1 and stored at 20°C.
Immediately before use, samples of dissolved peptide were thawed and diluted
to final bath concentrations
(109107 mol l1)
in physiological saline. During all experiments, the saline temperature was
cooled with an ice bath and regulated to within a few tenths of a degree at a
temperature of approximately 10°C.
Excitatory junctional potential recordings
Measurements of excitatory junctional potentials (EJPs) were made from the
gastric mill 4 (gm4), gastric mill 5a (gm5a), gastric mill 6a (gm6a), gastric
mill 8a (gm8a), pyloric 1 (p1) and pyloric 2 (p2) muscles using conventional 2
mol l1 potassium acetate-filled microelectrodes with
resistances of 710 M. The gm4 muscle is innervated by the dorsal
gastric (DG) neuron via the dorsal gastric nerve (dgn). The
gm5a muscle is innervated by the inferior cardiac (IC) neuron via the
medial ventricular nerve (mvn). The other four muscles are innervated
via the lateral ventricular nerve (lvn); gm6a and gm8a by
the lateral gastric (LG) neuron, p1 by the lateral pyloric (LP) neuron, and p2
by the pyloric (PY) neurons (Maynard and
Dando, 1974
; Selverston and
Moulins, 1987
; Weimann et
al., 1991
; Harris-Warrick et
al., 1992
). Innervating nerves were stimulated extracellularly
via stainless steel pin electrodes driven by an A-M systems Model
2100 isolated pulse stimulator (A-M Systems, Carlsborg, WA, USA). EJPs were
measured with an Axoclamp 2-B intracellular amplifier (Axon Instruments, Union
City, CA, USA), amplified 10-fold using a Model 440 instrumentation amplifier
(Brownlee Precision, San Jose, CA, USA) and recorded using a Digidata 1322A
acquisition system (Axon Instruments). EJP amplitude was defined as the peak
membrane potential of the EJP relative to the baseline potential prior to
stimulation. EJP amplitudes were analyzed using routines written in Matlab
(The MathWorks, Natick, MA, USA).
Contraction measurements
Recordings of contractions from the gm8 muscle were obtained using a FT03
force displacement transducer (Astro-Med, West Warwick, RI, USA). The
neuromuscular preparation consisted of fibers of both the gm8a and gm8b
muscles, which were not separated in order to minimize damage to the muscle
fibers and innervating nerves. One of the muscle insertions was pinned down to
the Sylgard in the recording dish, while the other insertion was tied to the
transducer with a short piece (3 cm) of size 6/0 silk suture thread (Fine
Science Tools, Foster City, CA, USA). The transducer was positioned so that
the muscle was stretched just past its relaxed length. The innervating nerve
was stimulated with a train of pulses applied through the pin electrode. This
resulted in muscle shortening, and the force transducer measured the increased
tension. The transducer signal was amplified by a factor of 10 000 using the
Brownlee Precision Model 440 amplifier and recorded using the Digidata
interface board. Contraction amplitudes were analyzed using the Clampfit
program (Axon Instruments).
Statistics
In the experiments in which the effect of a single concentration
(107 mol l1) of CabTRP Ia on the EJP
amplitude or contraction of a muscle was tested, a paired t-test was
used to test for statistical significance. Error bars on plots correspond to
standard errors.
Figure production
Anatomy figures were produced using a combination of Photoshop (version
7.0; Adobe Systems Inc., San Jose, CA, USA) and Canvas (version 8.0; Deneba
Systems Inc., Miami, FL, USA) software. Contrast and brightness were adjusted
as needed to optimize the clarity of the printed images. Mass spectra were
collected using IonSpec99 version 7.0 software (IonSpec Corp.). Boston
University Data Analysis (BUDA) version 1.4 was used to export the spectra as
a bitmap into Macromedia Fireworks MX 2004 Version 7.0 (Macromedia
Incorporated, San Francisco, CA, USA). Resolution was increased and peaks were
labeled with mass and peptide identity in Fireworks. Physiology figures were
produced using Sigma Plot (version 8.0; Systat Software, Point Richmond, CA,
USA).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Within the STNS of C. productus, numerous TRP-immunopositive
profiles were identified (Fig.
1). As with the previously mapped crab and lobster species
(Goldberg et al., 1988), the
most striking of these structures is a club-shaped plexus located in the
anterior medial portion of each CoG (Figs
1,
2; N=148 ganglia).
Labeling in the plexus consisted of a dense aggregation of nerve terminals
that originate from a fascicle of small (<1 µm)-diameter axons
projecting from the coc connecting the CoG to the thoracic nervous
system (Fig. 2A,B). On average,
the individual terminals that comprise the plexus were rather elongate, with
major cross-sectional diameters of <1 to >15 µm. In the posterior
portion of the plexus, the terminal aggregates were often extensively
fenestrated by unstained tubular areas
(Fig. 2C).
|
Neuroendocrine organs
In addition to examining the STNS for the presence of TRP-immunopositive
profiles, we also examined two well-known neuroendocrine organs located
outside this system, namely the SG and PO, for TRP-like labeling. In
brachyuran crabs, the SG is recognizable as a superficial, intensely
iridescent structure located between the medulla interna and medulla externa
of the eyestalk ganglia (Cooke and
Sullivan, 1982; Beltz,
1988
; Fingerman,
1992
). The PO is located on the dorsolateral wall of the
pericardial chamber and, in brachyurans, consists of two or more longitudinal
nerve trunks that are interconnected by vertical nerve bars
(Cooke and Sullivan, 1982
;
Beltz, 1988
;
Fingerman, 1992
). In neither
the SG (N=6) nor the PO (N=6) was any TRP immunoreactivity
evident (data not shown).
Distribution of hemolymph lacunae in the CoG
The existence of hemolymph sinuses and lacunae penetrating deep into the
ganglia of crustaceans is well documented (Abbott,
1971,
1972
;
Skinner, 1985
;
McGaw and Reiber, 2002
). In
the blue crab, Callinectes sapidus, mapping by radiography and
corrosion casting showed that the CoGs are among the ganglia fenestrated by
hemolymph lacunae (McGaw and Reiber,
2002
). As just described, the TRP-like labeling in the posterior
portion of the C. productus CoG plexus is fenestrated by tubular
unlabeled areas. One explanation for these regions of immunolabel avoidance is
that they are hemolymph lacunae. To investigate this hypothesis, we injected
physiological saline containing India ink into the pericardial chamber
surrounding the heart and allowed it to be pumped throughout the vascular
system, thereby filling hemolymph vessels with an easily visualized
substrate.
As can be seen in the ganglion shown in Fig. 3, numerous ink-filled lacunae are present in the CoG. While the location, size, shape and depth of invagination of the individual lacunae varied between ganglia, each CoG taken from an ink-injected animal (N=14 ganglia) possessed some deeply invaginated channels in its anterior medial quadrant, particularly in the posterior portion of this region.
|
|
Immunohistochemical survey for co-transmitters in the ACO
Previous work has shown that most crustacean neuroendocrine sites contain
large and often complex complements of signaling molecules
(Christie et al., 1995a; Li et
al., 2002a
,
2003
; Fu et al.,
2005a
,b
).
In an attempt to determine what other substances might be co-localized with
TRP in the ACO, we immunolabeled CoGs with antibodies generated against a
number of known crustacean hormones and/or neuromodulators and examined them
for labeling of the plexus. In some preparations, double-labeling with
substance P antibody was undertaken to unambiguously delimit the location of
the ACO. The assayed substances were the small molecule transmitter GABA, the
amines dopamine, histamine and serotonin, the gases carbon monoxide and nitric
oxide as well as the peptides allatostatin, buccalin, CCK, corazonin, CCAP,
FLRFamide, myomodulin, orcokinin, ß-PDH, proctolin and RPCH. While each
of the antibodies labeled neuronal profiles in the CoG and/or other regions of
the C. productus nervous system (data not shown), none gave rise to
labeling within the ACO (N
6 ganglia for each antibody;
Fig. 5).
|
Interestingly, while no co-transmitters were found in the ACO, our double-label experiments did show that this structure is surrounded in three dimensions by neuropil containing many other neuroactive compounds (Fig. 5). In many areas, these immunopositive neuropils directly abut ACO terminals. Thus, in addition to neuroendocrine release, the ACO may also function to modulate the CoG neuropil in a paracrine fashion.
Mass spectrometric detection of authentic CabTRP Ia in the CoG and hemolymph
Anterior medial quadrant of the CoG
As shown earlier, the ACO is immunolabeled by an antibody generated against
the vertebrate tachykinin substance P. In crustaceans, the only TRP thus far
identified is APSGFLGMRamide, commonly referred to as CabTRP Ia
(Christie et al., 1997a;
Nieto et al., 1998
;
Li et al., 2002a
;
Huybrechts et al., 2003
;
Yasuda-Kamatani and Yasuda,
2004
). Previous studies have shown that the substance P antibody
cross-reacts with this peptide (Christie
et al., 1997a
); thus, it is likely that the substance P
immunoreactivity seen in the ACO results from the presence of authentic CabTRP
Ia in this structure. To confirm that this is indeed the case, we isolated the
quadrant of the CoG containing the ACO and subjected it to direct tissue
MALDI-FTMS analysis.
As the mass spectrum in Fig. 6A shows, many peptides are present in the portion of the CoG containing the ACO. Among the detected peaks are ones corresponding to a number of previously identified peptides including several isoforms of orcokinins, corazonin, three FLRFamide-related peptides, Gly1-SIFamide and RPCH. Of particular interest is the mass/charge (m/z) peak at 934.501, which is essentially identical to the theoretical m/z of authentic CabTRP Ia (i.e. 934.493). This result is strongly suggestive of CabTRP Ia being present in authentic form in the anterior medial quadrant of the CoG and hence in the ACO.
|
Hemolymph
Since the ACO possesses the organization of a neuroendocrine organ, it is
logical to expect that the CabTRP Ia contained within it would be released
into the circulatory system. As none of the other known neuroendocrine organs
in C. productus (i.e. the SG, the PO and the ACP) exhibits any
evidence of TRP immunoreactivity, the ACO may well be the sole source of this
peptide in the hemolymph. To determine if CabTRP Ia does circulate, we
collected hemolymph samples from three crabs and subjected the extract to
MALDI-FTMS analysis. In two of three animals, the mass spectra of the
hemolymph extract showed an m/z peak corresponding to that
of authentic CabTRP Ia (i.e. m/z 934.491 in
Fig. 6B). This finding strongly
supports the notion that this peptide is a circulating hormone potentially
released from the ACO.
Physiological effects of CabTRP Ia on the muscles of the gastric mill and pylorus
In the crab C. borealis, Jorge-Rivera and colleagues showed that
many peptides that do not directly innervate the muscles of the gastric mill
and pyloric regions of the foregut are nonetheless potent modulators of the
musculature (Jorge-Rivera and Marder,
1996,
1997
;
Jorge-Rivera, 1997
;
Jorge-Rivera et al., 1998
). As
shown by our schematic diagram of substance P immunolabeling in the STNS, no
direct TRP innervation of the gastric mill or pyloric muscles is present in
C. productus (Fig. 1).
To determine if circulating CabTRP Ia can similarly modulate stomatogastric
muscles in C. productus, we studied its actions on EJP and
contraction amplitude for a subset of the intrinsic muscles of the
foregut.
Modulation of EJP amplitude
We tested the effect of 107 mol l1
CabTRP Ia on the amplitude of nerve-evoked EJPs in six stomatogastric muscles
(Fig. 7). Three of the muscles
(gm4, gm6a and gm8a) are innervated by motor neurons (i.e. DG or LG) involved
in generating the gastric mill motor pattern, while the other three muscles
(gm5a, p1 and p2) are innervated by neurons (IC, LP or PY, respectively) that
participate in the pyloric circuit
(Maynard and Dando, 1974;
Selverston and Moulins, 1987
;
Weimann et al., 1991
;
Harris-Warrick et al., 1992
).
For all muscles, a statistically significant and reversible increase in EJP
amplitude was noted (gm4, N=5; gm5a, N=6; gm6a,
N=8; gm8a, N=6; p1, N=8; p2, N=6). For all
muscles, the maximum effect of CabTRP Ia occurred approximately 510 min
after bath application was initiated and required at least 30 min of rinsing
in physiological saline to return to baseline. No effect of CabTRP Ia on
membrane potential was observed for any of the muscles tested.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we have shown that the ACO of C. productus
consists of aggregations of tachykinin-immunopositive nerve terminals that
directly abut/surround hemolymph lacunae. Thus, the general organization of
this structure is homologous to those of other crustacean neuroendocrine
sites, and to the SG in particular. Based on this homology, we believe that,
like the SG, the ACO is a neuroendocrine release organ. Our identification of
the ACO as a neuroendocrine center now brings the number of characterized
neuroendocrine organs in C. productus to four (i.e. the SG, PO, ACP
and ACO; Christie et al.,
2004a; Christie and Messinger,
2005
; Fu et al.,
2005a
,b
;
Messinger et al., 2005
).
Given that structures apparently homologous to the C. productus
ACO have been identified in species from six infraorders of decapods [i.e.
Brachyura (true crabs), Anomura (hermit crabs, porcelain crabs and squat
lobsters), Palinura (spiny lobsters), Thalassinidea (ghost shrimp), Astacidea
(chelate lobsters and freshwater crayfish) and Caridea (caridean shrimp);
Goldberg et al., 1988;
Messinger et al., 2004
], the
ACO appears to be a highly conserved structure and we hypothesize that its
function as a neuroendocrine center is conserved in other decapod species.
The hormone complement of the ACO appears to be limited in comparison to those of other neuroendocrine sites
Recently, much work has focused on the identification of hormone
complements in the neuroendocrine organs (i.e. the SG, PO and ACP) of C.
productus (Hsu et al.,
2004,
2005a
,b
;
de la Iglesia et al., 2005
; Fu
et al.,
2005a
,b
;
Messinger et al., 2005
). Like
those of other crustacean species
(Christie et al., 1995a
; Li et
al., 2002a
,
2003
), each of these sites has
been shown to contain multiple neuroactive compounds. For example, using a
combination of anatomical, mass spectrometric and molecular techniques, over
50 peptide hormones have been identified from the C. productus SG
(Hsu et al., 2004
,
2005a
,b
;
de la Iglesia et al., 2005
; Fu
et al.,
2005a
,b
).
Similarly, over 40 putative hormones (peptides, amines and small molecules)
have been identified in the C. productus PO
(Hsu et al., 2004
;
Fu et al., 2005b
). Even in the
ACP, which is innervated by just four neurons, multiple hormones have been
identified (Christie et al.,
2004a
; Hsu et al.,
2004
; Christie and Messinger,
2005
; Messinger et al.,
2005
).
In contrast to the chemical complexity seen in the hormone complements of the other neuroendocrine organs of C. productus, only CabTRP Ia has been identified as a putative hormone in the ACO. Immunohistochemistry using antibodies to other known crustacean hormones/neuromodulators failed to identify any co-transmitters in this site. However, in our mass spectrometric characterization of the anterior medial quadrant of the CoG that contains the ACO, we detected a number of peptides for which no antibodies are currently available (e.g. Gly1-SIFamide and HLGSLYRamide) as well as a number of currently unidentified ones, and it is certainly possible that some of these peptides are contained within and utilized as signaling agents by the ACO.
The ACO as a neurohemal source of circulating CabTRP Ia
Thus far, the only neuroactive compound identified in the C.
productus ACO is CabTRP Ia. In our study, we show via mass
spectrometry that this peptide is also present in the hemolymph of this crab.
Using immunohistochemistry, we screened the other known neuroendocrine organs
of C. productus (i.e. the SG, PO and ACP) for the presence of CabTRP
Ia and found that each tissue lacked labeling. Moreover, mass spectrometric
analysis also failed to detect this peptide in these sites
(Christie and Messinger, 2005;
Fu et al., 2005b
;
Messinger et al., 2005
).
Collectively, these findings suggest that if the CabTRP Ia seen in the
hemolymph is derived from a neuroendocrine organ, the ACO is a likely source
of the peptide.
Hormonally delivered CabTRP Ia is capable of modulating the contractile properties of the musculature of the foregut
As just described, CabTRP Ia is detectable in the hemolymph of C.
productus, and the ACO is one potential source of this circulating
peptide. Given a hormonal mode of delivery, multiple target tissues are likely
to exist for this peptide in the crab. As we have shown here, one target of
hemolymph-borne CabTRP Ia is the musculature of the foregut. In this species,
none of the muscles of the gastric mill or pyloric regions of the foregut is
directly innervated by TRP-immunopositive axons. Nevertheless, a hormonally
relevant concentration (107 mol l1) of
CabTRP Ia increased EJPs in three of three gastric mill and three of three
pyloric muscles tested. Moreover, concentrations as low as
108 mol l1 were observed to consistently
increase the contraction amplitude of at least one muscle, with the
enhancement presumably due in part to the CabTRP Ia-evoked increase in EJP
amplitude.
The most thoroughly studied peptide modulators of stomatogastric
musculature are TNRNFLRFamide and SDRNFLRFamide in the crab C.
borealis (Jorge-Rivera and Marder,
1996; Jorge-Rivera et al.,
1998
). Our investigation of the effects of CabTRP Ia in C.
productus, while not nearly as extensive as the earlier work, has
produced some similar observations. Application of either FLRFamide peptide
increased the nerve-evoked contraction in 15 out of 17 muscles tested, with a
threshold concentration of 1010 mol l1
(TNRNFLRFamide) or 109108 mol
l1 (SDRNFLRFamide)
(Jorge-Rivera and Marder,
1996
). We measured the threshold concentration of CabTRP Ia for an
increase on gm8 contraction to be between 109 mol
l1 and 108 mol l1 and
observed effects on EJP amplitude in all six muscles tested.
Although we have not explored the biophysical mechanisms responsible for
the increase in EJP amplitude, we note that no shift in muscle membrane
potential was observed that would clearly indicate a change in muscle input
resistance. In the crab C. borealis, CabTRP Ia and four other
peptides (including TNRNFLRFamide) have been observed to activate an inward
current (Swensen and Marder,
2000) in subsets of STG motor neurons
(Swensen and Marder, 2001
).
Interestingly, the other four peptides, all presumably acting as hormones,
also increase muscle contractions in C. borealis
(Jorge-Rivera et al., 1998
).
Given the low-frequency motor discharge that drives many of the foregut
muscles, we hypothesize that circulating CabTRP Ia may be crucial for
maintaining appreciable muscle contractions in the gastric mill and pylorus,
as has been proposed for other peptide hormones, including the extended
FLRFamides (Jorge-Rivera and Marder,
1996
).
Paracrine signaling from the ACO to intrinsic targets in the CoG
Our results show that many of the nerve terminals constituting the ACO
envelop and/or directly abut hemolymph lacunae. This organization should allow
released hormone direct access to the circulatory system and is widely
recognized as the defining characteristic of a crustacean neuroendocrine
release site (Cooke and Sullivan,
1982; Beltz, 1988
;
Fingerman, 1992
;
Christie et al., 2004a
).
Intriguingly, there are some areas of the ACO that do not directly abut the
circulatory system, but instead are in apposition to putative synaptic
neuropil. Here, peptide released from the ACO may also function as a paracrine
modulator of intrinsic CoG targets.
One possible role for paracrine signaling by the ACO is the coordination of
multiple neuroendocrine systems (Fig.
9). The soma of the large (L)-cell, which provides both
peptidergic and aminergic innervation to the PO
(Cooke and Sullivan, 1982;
Beltz, 1988
;
Fingerman, 1992
), is located
within the CoG and, in C. productus, extends processes to the general
vicinity of the ACO (A.E.C., unpublished observations). Likewise, the somata
innervating the ACP [i.e. anterior commissural neurons 1 and 2 (ACN1/2);
Christie et al., 2004a
;
Christie and Messinger, 2005
;
Messinger et al., 2005
] are
contained within the CoG and arborize in the vicinity of the plexus. If the
L-cell and/or ACN1/2 are modulated by CabTRP Ia (or some other unidentified
co-transmitter within the ACO), then elements of at least two other
neuroendocrine centers (i.e. the PO and ACP) could be simultaneously
modulated/synchronized locally within the CoG, with concurrent release of
hormones to the circulatory system.
|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abbott, N. J. (1971). The organization of the cerebral ganglion in the shore crab, Carcinus maenas. II. The relation of intracerebral blood vessels to other brain elements. Z. Zellforsch. Mikrosk. Anat. 120,401 -419.[CrossRef]
Abbott, N. J. (1972). Access of ferritin to the interstitial space of Carcinus brain from intracerebral blood vessels. Tissue Cell. 4,99 -104.[Medline]
Bartos, M. and Nusbaum, M. P. (1997).
Intercircuit control of motor pattern modulation by presynaptic inhibition.
J. Neurosci. 17,2247
-2256.
Beenhakker, M. P., Blitz, D. M. and Nusbaum, M. P.
(2004). Long-lasting activation of rhythmic neuronal activity by
a novel mechanosensory system in the crustacean stomatogastric nervous system.
J. Neurophysiol. 91,78
-91.
Beltz, B. S. (1988). Crustacean neurohormones. In Invertebrate Endocrinolog, Vol. 2, Endocrinology of Selected Invertebrate Types (ed. H. Laufer, R. G. H. Downer), pp. 235-258. New York: Alan R. Liss, Inc.
Blitz, D. M., Christie, A. E., Marder, E. and Nusbaum, M. P. (1995). Distribution and effects of tachykinin-like peptides in the stomatogastric nervous system of the crab, Cancer borealis. J. Comp. Neurol. 354,282 -294.[CrossRef][Medline]
Blitz, D. M., Christie, A. E., Coleman, M. J., Norris, B. J.,
Marder, E. and Nusbaum, M. P. (1999). Different
proctolin neurons elicit distinct motor patterns from a multifunctional
neuronal network. J. Neurosci.
19,5449
-5463.
Bungart, D., Dircksen, H. and Keller, R. (1994). Quantitative determination and distribution of the myotropic neuropeptide orcokinin in the nervous system of astacidean crustaceans. Peptides 15,393 -400.[CrossRef][Medline]
Cerri, P. S., de Faria, F. P., Villa, R. G. and Katchburian, E. (2004). Light microscopy and computer three-dimensional reconstruction of the blood capillaries of the enamel organ of rat molar tooth germs. J. Anat. 204,191 -195.[CrossRef][Medline]
Chang, M. M., Leeman, S. E. and Niall, H. D. (1971). Amino-acid sequence of substance P. Nat. New. Biol. 232,86 -87.[Medline]
Christie, A. E. and Messinger, D. I. (2005). Hormonal signaling from the crustacean stomatogastric nervous system. Comp. Biochem. Physiol. 144A, S157.
Christie, A. E. and Nusbaum, M. P. (1995). Distribution and effects of corazonin-like and allatotropin-like peptides in the crab stomatogastric nervous system. Soc. Neurosci. Abstr. 21,629 .
Christie, A. E., Hall, C., Oshinsky, M. and Marder, E.
(1994). Buccalin-like and myomodulin-like peptides in the
stomatogastric ganglion of the crab Cancer borealis. J. Exp.
Biol. 193,337
-343.
Christie, A. E., Skiebe, P. and Marder, E. (1995a). Matrix of neuromodulators in neurosecretory structures of the crab Cancer borealis. J. Exp. Biol. 198,2431 -2439.[Medline]
Christie, A. E., Baldwin, D., Turrigiano, G., Graubard, K. and Marder, E. (1995b). Immunocytochemical localization of multiple cholecystokinin-like peptides in the stomatogastric nervous system of the crab Cancer borealis. J. Exp. Biol. 198,263 -271.[Medline]
Christie, A. E., Lundquist, C. T., Nässel, D. R. and
Nusbaum, M. P. (1997a). Two novel tachykinin-related peptides
from the nervous system of the crab Cancer borealis. J. Exp.
Biol. 200,2279
-2294.
Christie, A. E., Baldwin, D. H., Marder, E. and Graubard, K. (1997b). Organization of the stomatogastric neuropil of the crab, Cancer borealis, as revealed by modulator immunocytochemistry. Cell Tissue Res. 288,135 -148.[CrossRef][Medline]
Christie, A. E., Edwards, J. M., Cherny, E., Clason, T. A. and Graubard, K. (2003). Immunocytochemical evidence for nitric oxide- and carbon monoxide-producing neurons in the stomatogastric nervous system of the crayfish Cherax quadricarinatus. J. Comp. Neurol. 467,293 -306.[CrossRef][Medline]
Christie, A. E., Cain, S. D., Edwards, J. M., Clason, T. A.,
Cherny, E., Lin, M., Manhas, A. S., Sellereit, K. L., Cowan, N. G.,
Nold, K. A. et al. (2004a). The anterior cardiac plexus: an
intrinsic neurosecretory site within the stomatogastric nervous system of the
crab Cancer productus. J. Exp. Biol.
207,1163
-1182.
Christie, A. E., Stein, W., Quinlan, J. E., Beenhakker, M. P., Marder, E. and Nusbaum, M. P. (2004b). Actions of a histaminergic/peptidergic projection neuron on rhythmic motor patterns in the stomatogastric nervous system of the crab Cancer borealis. J. Comp. Neurol. 469,153 -169.[CrossRef][Medline]
Coleman, M. J. and Nusbaum, M. P. (1994). Functional consequences of compartmentalization of synaptic input. J. Neurosci. 14,6544 -6552.[Abstract]
Coleman, M. J., Meyrand, P. and Nusbaum, M. P. (1995). A switch between two modes of synaptic transmission mediated by presynaptic inhibition. Nature 378,502 -505.[CrossRef][Medline]
Cooke, I. M. and Sullivan, R. E. (1982). Hormones and neurosecretion. In The Biology of Crustacea, Vol. 3, Neurobiology: Structure and Function (ed. H. L. Atwood and D. C. Sandeman), pp. 206-278. New York: Academic Press.
Cuello, A. C., Galfre, G. and Milstein, C.
(1979). Detection of substance P in the central nervous system by
a monoclonal antibody. Proc. Natl. Acad. Sci. U.S.A.
76,3532
-3536.
de la Iglesia, H. O., Hsu, Y. A., Weller, J. R. and Christie, A. E. (2005). Molecular cloning of cDNAs encoding crustacean hyperglycemic hormone (CHH) and its precursor related peptide (CPRP) from the sinus gland of the crab, Cancer productus. Program No. 30.6. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience 2005 (Online).
Dircksen, H. and Keller, H. (1988). Immunocytochemical localization of CCAP, a novel crustacean cardioactive peptide, in the nervous system of the shore crab Carcinus maenas L. Cell Tissue Res. 254,347 -360.
Dircksen, H., Zahnow, C. A., Gaus, G., Keller, R., Rao, K. R. and Riehm, J. P. (1987). The ultrastructure of nerve endings containing pigment dispersing hormone (PDH) in the crustacean sinus glands: identification by an antiserum against a synthetic PDH. Cell Tissue Res. 250,377 -387.
Erspamer, V. and Anastasi, A. (1962). Structure and pharmacological actions of eledoisin, the active endecapeptide of the posterior salivary glands of Eledone. Experientia 18, 58-59.[Medline]
Farley, R. D. (1990). Regulation of air and blood flow through the book lungs of the desert scorpion Paruroctonus mesaensis. Tissue Cell 22,547 -569.[CrossRef]
Fénelon, V. S., Kilman, V., Meyrand, P. and Marder, E. (1999). Sequential developmental acquisition of neuromodulatory inputs to a central pattern-generating network. J. Comp. Neurol. 408,335 -351.[CrossRef][Medline]
Fingerman, M. (1992). Decapod crustacean glands. In Microscopic Anatomy of Invertebrates, Vol. 10, Decapod Crustacea (ed. F. W. Harrison and A. G. Humes), pp.345 -394. New York: Wiley-Liss Inc.
Fingerman, M., Hanumante, M. M., Kulkarni, G. K., Ikeda, R. and Vacca, L. L. (1985). Localization of substance P-like, leucine-enkephalin-like, methionine-enkephalin-like, and FMRFamide-like immunoreactivity in the eyestalk of the fiddler crab, Uca pugilator.Cell Tissue Res. 241,473 -477.[Medline]
Fu, Q., Christie, A. E. and Li, L. (2005a). Mass spectrometric characterization of crustacean hyperglycemic hormone precursor-related peptides (CPRPs) from the sinus gland of the crab, Cancer productus. Peptides In press.
Fu, Q., Kutz, K. K., Schmidt, J. J., Hsu, Y. A., Messinger, D. I., Cain, S. D., de la Iglesia, H. O., Christie, A. E. and Li, L. (2005b). Hormone complement of the Cancer productus sinus gland and pericardial organ: an anatomical and mass spectrometric investigation. J. Comp. Neurol. In press.
Glantz. R. M., Miller, C. S. and Nässel, D. R.
(2000). Tachykinin-related peptide and GABA-mediated presynaptic
inhibition of crayfish photoreceptors. J. Neurosci.
20,1780
-1790.
Goldberg, D., Nusbaum, M. P. and Marder, E. (1988). Substance P-like immunoreactivity in the stomatogastric nervous systems of the crab Cancer borealis and the lobsters Panulirus interruptus and Homarus americanus. Cell Tissue Res. 252,515 -522.[CrossRef][Medline]
Grivas, I., Michaloudi, H., Batzios, C., Chiotelli, M., Papatheodoropoulos, C., Kostopoulos, G. and Papadopoulos, G. C. (2003). Vascular network of the rat hippocampus is not homogeneous along the septotemporal axis. Brain Res. 971,245 -249.[CrossRef][Medline]
Harris-Warrick, R. M., Marder, E., Selverston, A. I. and Moulins, M. (ed.) (1992). Dynamic Biological Networks: The Stomatogastric Nervous System. Cambridge: MIT Press.
Hogers, B., DeRuiter, M. C., Baasten, A. M., Gittenberger-de
Groot, A. C. and Poelmann, R. E. (1995). Intracardiac
blood flow patterns related to the yolk sac circulation of the chick embryo.
Circ. Res. 76,871
-877.
Hsu, Y. A., Weller, J. R., de la Iglesia, H. O. and Christie, A. E. (2004). Immunocytochemical evidence for crustacean hyperglycemic hormone-related peptides in the neuroendocrine organs of Cancer crabs. Program No. 274.4. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience 2004 (Online).
Hsu, Y. A., de la Iglesia, H. O. and Christie, A. E. (2005a). Crustacean hyperglycemic hormone-related peptides in Cancer productus. Comp. Biochem. Physiol. 141A, S157.
Hsu, Y. A., Messinger, D. I., Christie, A. E. and de la Iglesia, H. O. (2005b). Molecular characterization and putative circadian cycling of ß-pigment dispersing hormone (ß-PDH) in the sinus gland of the crab Cancer productus. Program No. 30.3. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience 2005 (Online).
Hutchinson, D. A. and Savitzky, A. H. (2004). Vasculature of the parotoid glands of four species of toads (bufonidae: bufo). J. Morphol. 260,247 -254.[CrossRef][Medline]
Huybrechts, J., Nusbaum, M. P., Bosch, L. V., Baggerman, G., De Loof, A. and Schoofs, L. (2003). Neuropeptidomic analysis of the brain and thoracic ganglion from the Jonah crab, Cancer borealis. Biochem. Biophys. Res. Commun. 308,535 -544.[CrossRef][Medline]
Johnson, E. C., Garczynski, S. F., Park, D., Crim, J. W.,
Nässel, D. R. and Taghert, P. H. (2003).
Identification and characterization of a G protein-coupled receptor for the
neuropeptide proctolin in Drosophila melanogaster. Proc. Natl.
Acad. Sci. USA 100,6198
-6203.
Jorge-Rivera, J. C. (1997). Modulation of stomatogastric musculature in the crab, Cancer borealis. PhD dissertation, Brandeis University, Waltham.
Jorge-Rivera, J. C. and Marder, E. (1996). TNRNFLRFamide and SDRNFLRFamide modulate muscles of the stomatogastric system of the crab Cancer borealis. J. Comp. Physiol. A. 179,741 -751.[Medline]
Jorge-Rivera, J. C. and Marder, E. (1997).
Allatostatin decreases stomatogastric neuromuscular transmission in the crab
Cancer borealis. J. Exp. Biol.
200,2937
-2946.
Jorge-Rivera, J. C., Sen, K., Birmingham, J. T., Abbott, L. F.
and Marder, E. (1998). Temporal dynamics of convergent
modulation at a crustacean neuromuscular junction. J.
Neurophysiol. 80,2559
-2570.
Kutz, K. K., Schmidt, J. J. and Li, L. (2004). In situ tissue analysis of neuropeptides by MALDI FTMS in-cell accumulation. Anal. Chem. 76,5630 -5640.[CrossRef][Medline]
Lane, N. J., Harrison, J. B. and Bowerman, R. F. (1981). A vertebrate-like bloodbrain barrier, with intraganglionic blood channels and occluding junctions, in the scorpion. Tissue Cell 13,557 -576.[CrossRef][Medline]
Langworthy, K., Helluy, S., Benton, J. and Beltz, B. (1997). Amines and peptides in the brain of the American lobster: immunocytochemical localization patterns and implications for brain function. Cell Tissue Res. 288,191 -206.[CrossRef][Medline]
Li, L., Pulver, S. R., Kelley, W. P., Sweedler, J. V. and Marder, E. (2002a). Towards revealing the full complement of neurohormones in crustacean pericardial organs and hemolymph by mass spectrometry. Program No. 609.13. 2002 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2002 (Online).
Li, L., Pulver, S. R., Kelley, W. P., Thirumalai, V., Sweedler, J. V. and Marder, E. (2002b). Orcokinin peptides in developing and adult crustacean stomatogastric nervous systems and pericardial organs. J. Comp. Neurol. 444,227 -244.[CrossRef][Medline]
Li, L., Kelley, W. P., Billimoria, C. P., Christie, A. E., Pulver, S. R., Sweedler, J. V. and Marder, E. (2003). Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis. J. Neurochem. 87,642 -656.[CrossRef][Medline]
Madsen, A. J., Herman, W. S. and Elde, R. (1985). Differential distribution of two homologous neuropeptides (RPCH & AKH) in the crayfish nervous system. Soc. Neurosci. Abstr. 11,941 .
Mancillas, J. R., McGinty, J. F., Selverston, A. I., Karten, H. and Bloom, F. E. (1981). Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurones of lobster. Nature 293,576 -578.[CrossRef][Medline]
Marinkovic, S., Milisavljevic, M., Gibo, H., Malikovic, A. and Djulejic, V. (2004). Microsurgical anatomy of the perforating branches of the vertebral artery. Surg. Neurol. 61,190 -197.[CrossRef][Medline]
Maynard, D. M. and Dando, M. R. (1974). The structure of the stomatogastric neuromuscular system in Callinectes sapidus, Homarus americanus and Panulirus argus (Decapoda Crustacea). Phil. Trans. R. Soc. Lond. B 268,161 -220.[Medline]
McGaw, I. J. and Reiber, C. L. (2002). Cardiovascular system of the blue crab Callinectes sapidus. J. Morphol. 251,1 -21.[CrossRef][Medline]
Meola, S. M., Clottens, F. L., Holman, G. M., Nachman, R. J., Nichols, R., Schoofs, L., Wright, M. S., Olson, J. K., Hayes, T. K. and Pendleton, M. W. (1998). Isolation and immunocytochemical characterization of three tachykinin-related peptides from the mosquito, Culex salinarius. Neurochem. Res. 23,189 -202.[CrossRef][Medline]
Messinger, D. I., Ngo, C. T., Cain, S. D. and Christie, A. E. (2004). Phylogenetic conservation of a tachykinin-containing neuroendocrine organ in the commissural ganglia of decapod crustaceans. Program No. 274.3. 2004 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience, 2004 (Online).
Messinger, D. I., Verley, D. R., Fu, Q., Kutz, K. K., Hsu, Y. A., Li, L., Birmingham, J. T. and Christie, A. E. (2005). Structural and functional characterization of the anterior cardiac neuron 1/2-anterior cardiac plexus (ACN1/2-ACP) neuroendocrine system of the crab Cancer productus. Soc. Neurosci. Abstr. Program No. 30.8. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience 2005 (Online).
Miller, M. W., Alevizos, A., Cropper, E. C., Vilim, F. S., Karagogeos, D., Kupfermann, I. and Weiss, K. R. (1991). Localization of myomodulin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia californica.J. Comp. Neurol. 314,627 -644.[CrossRef][Medline]
Miller, M. W., Alevizos, A., Cropper, E. C., Kupfermann, I. and Weiss, K. R. (1992). Distribution of buccalin-like immunoreactivity in the central nervous system and peripheral tissues of Aplysia californica. J. Comp. Neurol. 320,182 -195.[CrossRef][Medline]
Mortin, L. I. and Marder, E. (1991). Differential distribution of beta-pigment-dispersing hormone (beta-PDH)-like immunoreactivity in the stomatogastric nervous system of five species of decapod crustaceans. Cell Tissue Res. 265, 19-33.[CrossRef][Medline]
Muren, J. E. and Nässel, D. R. (1996). Radioimmunoassay determination of tachykinin-related peptide in different portions of the central nervous system and intestine of the cockroach Leucophaea maderae. Brain Res. 739,314 -321.[CrossRef][Medline]
Muren, J. E. and Nässel, D. R. (1997). Seven tachykinin-related peptides isolated from the brain of the Madeira cockroach: evidence for tissue-specific expression of isoforms. Peptides 18,7 -15.[CrossRef][Medline]
Murray, B. and Wilson, D. J. (1997). Muscle patterning, differentiation and vascularisation in the chick wing bud. J. Anat. 190,261 -273.[CrossRef][Medline]
Nachman, R. J., Moyna, G., Williams, H. J., Zabrocki, J.,
Zadina, J. E., Coast, G. M. and Vanden Broeck, J.
(1999). Comparison of active conformations of the
insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family
pairs. Ann. N.Y. Acad. Sci.
897,388
-400.
Nässel, D. R. (1993). Insect myotropic peptides: differential distribution of locustatachykinin- and leucokinin-like immunoreactive neurons in the locust brain. Cell Tissue Res. 274,27 -40.[CrossRef][Medline]
Nässel, D. R. (1999). Tachykinin-related peptides in invertebrates: a review. Peptides 20,141 -158.[CrossRef][Medline]
Nieto, J., Veelaert, D., Derua, R., Waelkens, E., Cerstiaens, A., Coast, G., Devreese, B., Van Beeumen, J., Calderon, J., De Loof, A. et al. (1998). Identification of one tachykinin- and two kinin-related peptides in the brain of the white shrimp, Penaeus vannamei. Biochem. Biophys. Res. Commun. 248,406 -411.[CrossRef][Medline]
Norris, B. J., Coleman, M. J. and Nusbaum, M. P.
(1994). Recruitment of a projection neuron determines gastric
mill motor pattern selection in the stomatogastric nervous system of the crab,
Cancer borealis. J. Neurophysiol.
72,1451
-1463.
Norris, B. J., Coleman, M. J. and Nusbaum, M. P.
(1996). Pyloric motor pattern modification by a newly identified
projection neuron in the crab stomatogastric nervous system. J.
Neurophysiol. 75,97
-108.
Nusbaum, M. P. (2002). Regulating peptidergic modulation of rhythmically active neural circuits. Brain. Behav. Evol. 60,378 -387.[CrossRef][Medline]
Nusbaum, M. P. and Marder, E. (1988). A neuronal role for a crustacean red pigment concentrating hormone-like peptide: neuromodulation of the pyloric rhythm in the crab, Cancer borealis.J. Exp. Biol. 135,165 -181.
Panula, P., Happola, O., Airaksinen, M. S., Auvinen, S. and Virkamaki, A. (1988). Carbodiimide as a tissue fixative in histamine immunohistochemistry and its application in developmental neurobiology. J. Histochem. Cytochem. 36,259 -269.[Abstract]
Pulver, S. R. and Marder, E. (2002). Neuromodulatory complement of the pericardial organs in the embryonic lobster, Homarus americanus. J. Comp. Neurol. 451, 79-90.[CrossRef][Medline]
Renkin, E. M. (1985). Flow and distribution of India ink in microvessels of the frog. Microvasc. Res. 29, 32-44.[CrossRef][Medline]
Renkin, E. M., Gray, S. D. and Dodd, L. R. (1981). Filling of microcirculation in skeletal muscles during timed India ink perfusion. Am. J. Physiol. 241,174 -186.
Sandeman, D. C., Sandeman, R. E. and de Couet, H. G. (1990a). Extraretinal photoreceptors in the brain of the crayfish Cherax destructor. J. Neurobiol. 21,619 -629.[CrossRef][Medline]
Sandeman, R. E., Sandeman, D. C. and Watson, A. H. (1990b). Substance P antibody reveals homologous neurons with axon terminals among somata in the crayfish and crab brain. J. Comp. Neurol. 294,569 -582.[CrossRef][Medline]
Sasaki, F., Doshita, A., Matsumoto, Y., Kuwahara, S., Tsukamoto, Y. and Ogawa, K. (2003). Embryonic development of the pituitary gland in the chick. Cells Tissues Organs 173, 65-74.[CrossRef][Medline]
Schmidt, M. (1997a). Distribution of presumptive chemosensory afferents with FMRFamide- or substance P-like immunoreactivity in decapod crustaceans. Brain Res. 746, 71-84.[CrossRef][Medline]
Schmidt, M. (1997b). Distribution of centrifugal neurons targeting the soma clusters of the olfactory midbrain among decapod crustaceans. Brain Res. 752, 15-25.[CrossRef][Medline]
Schmidt, M. and Ache, B. W. (1994). Descending neurons with dopamine-like or with substance P/FMRFamide-like immunoreactivity target the somata of olfactory interneurons in the brain of the spiny lobster, Panulirus argus. Cell Tissue Res. 278,337 -352.[CrossRef][Medline]
Schmidt, M. and Ache, B. W. (1997). Immunocytochemical analysis of glomerular regionalization and neuronal diversity in the olfactory deutocerebrum of the spiny lobster Cell Tissue Res. 287,541 -563.[CrossRef][Medline]
Schoofs, L., Holman, G. M., Hayes, T. K., Kochansky, J. P., Nachman, R. J. and De Loof, A. (1990a). Locustatachykinin III and IV: two additional insect neuropeptides with homology to peptides of the vertebrate tachykinin family. Regul. Pept. 31,199 -212.[CrossRef][Medline]
Schoofs, L., Holman, G. M., Hayes, T. K., Nachman, R. J. and De Loof, A. (1990b). Locustatachykinin I and II, two novel insect neuropeptides with homology to peptides of the vertebrate tachykinin family. FEBS Lett. 261,397 -401.[CrossRef][Medline]
Schoofs, L., Vanden Broeck, J. and De Loof, A. (1993). The myotropic peptides of Locusta migratoria: structures, distribution, functions and receptors. Insect Biochem. Mol. Biol. 23,859 -881.[CrossRef][Medline]
Selverston, A. I. and Moulins. M. (ed.) (1987). The Crustacean Stomatogastric System. Berlin: Springer.
Severini, C., Improta, G., Falconieri-Erspamer, G., Salvadori,
S. and Erspamer, V. (2002). The tachykinin peptide
family. Pharmacol. Rev.
54,285
-322.
Siviter, R. J., Coast, G. M., Winther, A. M., Nachman, R. J.,
Taylor, C. A., Shirras, A. D., Coates, D., Isaac, R. E. and
Nässel, D. R. (2000). Expression and functional
characterization of a Drosophila neuropeptide precursor with homology
to mammalian preprotachykinin A. J. Biol. Chem.
275,23273
-23280.
Skiebe, P., Dreger, M., Meseke, M., Evers, J. F. and Hucho, F. (2002). Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish, Cherax destructor.J. Comp. Neurol. 444,245 -259.[CrossRef][Medline]
Skinner, K. (1985). The structure of the fourth abdominal ganglion of the crayfish, Procambarus. clarki (Girard). I. Tracts in the ganglionic core. J. Comp. Neurol. 234,168 -181.[CrossRef][Medline]
Stangier, J., Hilbich, C., Dircksen, H. and Keller, R. (1988). Distribution of a novel cardioactive neuropeptide (CCAP) in the nervous system of the shore crab Carcinus maenas.Peptides 9,795 -800.[CrossRef][Medline]
Stay, B., Chan, K. K. and Woodhead, A. P. (1992). Allatostatin-immunoreactive neurons projecting to the corpora allata of adult Diploptera punctata. Cell Tissue Res. 270,15 -23.[CrossRef][Medline]
Swensen, A. M. and Marder, E. (2000). Multiple
peptides converge to activate the same voltage-dependent current in a central
pattern-generating circuit. J. Neurosci.
20,6752
-6759.
Swensen, A. M. and Marder, E. (2001).
Modulators with convergent cellular actions elicit distinct circuit outputs.
J. Neurosci. 21,4050
-4058.
Swensen, A. M., Golowasch, J., Christie, A. E., Coleman, M. J.,
Nusbaum, M. P. and Marder, E. (2000). GABA and
responses to GABA in the stomatogastric ganglion of the crab Cancer
borealis. J. Exp. Biol.
203,2075
-2092.
Takeuchi, H., Yasuda, A., Yasuda-Kamatani, Y., Kubo, T. and Nakajima, T. (2003). Identification of a tachykinin-related neuropeptide from the honeybee brain using direct MALDI-TOF MS and its gene expression in worker, queen and drone heads. Insect Mol. Biol. 12,291 -298.[CrossRef][Medline]
Thirumalai, V. and Marder, E. (2002).
Colocalized neuropeptides activate a central pattern generator by acting on
different circuit targets. J. Neurosci.
22,1874
-1882.
Tierney, A. J., Godleski, M. S. and Rattananont, P. (1999). Serotonin-like immunoreactivity in the stomatogastric nervous systems of crayfishes from four genera. Cell Tissue Res. 295,537 -551.[CrossRef][Medline]
Turrigiano, G. G. and Selverston, A. I. (1991). Distribution of cholecystokinin-like immunoreactivity within the stomatogastric nervous systems of four species of decapod crustacea. J. Comp. Neurol. 305,164 -176.[CrossRef][Medline]
Vanden Broeck, J., Torfs, H., Poels, J., Van Poyer, W., Swinnen,
E., Ferket, K. and De Loof, A. (1999). Tachykinin-like
peptides and their receptors. A review. Ann. N.Y. Acad.
Sci. 897,374
-387.
Veenstra, J. A. (1991). Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana. Peptides 12,1285 -1289.[CrossRef][Medline]
Weimann, J. M., Meyrand, P. and Marder, E.
(1991). Neurons that form multiple pattern generators:
identification and multiple activity patterns of gastric/pyloric neurons in
the crab stomatogastric system. J. Neurophysiol.
65,111
-122.
Winther, A. M. and Nässel, D. R. (2001).
Intestinal peptides as circulating hormones: release of tachykinin-related
peptide from the locust and cockroach midgut. J. Exp.
Biol. 204,1269
-1280.
Wood, D. E., Manor, Y., Nadim, F. and Nusbaum, M. P. (2004). Intercircuit control via rhythmic regulation of projection neuron activity. J. Neurosci. 24,7455 -7463.[CrossRef][Medline]
Woodhead, A. P., Stoltzman, C. A. and Stay, B. (1992). Allatostatins in the nerves of the antennal pulsatile organ muscle of the cockroach Diploptera punctata. Arch. Insect. Biochem. Physiol. 20,253 -263.[CrossRef][Medline]
Yasuda-Kamatani, Y. and Yasuda, A. (2004).
APSGFLGMRamide is a unique tachykinin-related peptide in crustaceans.
Eur. J. Biochem. 271,1546
-1556.