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Enhancement of muscle contraction in the stomach of the crab Cancer borealis: a possible hormonal role for GABA.


Although [gamma]-aminobutyric acid (GABA) is best known as the principal inhibitory neurotransmitter in the mammalian central nervous system, instances of excitatory GABAergic actions have been reported in a number of vertebrate and invertebrate systems. In many cases, these actions are mediated by the same ionotropic GAB[A.sub.A] receptor that normally causes inhibition, and excitation results from a depolarized C[l.sup.-] reversal potential or the flow of other anions through the channels (Alger and Nicoll, 1979; Andersen et al., 1980; Thalmann et al., 1981; Arakawa and Okada, 1988; Staley et al., 1995; Staley and Proctor, 1999; Ben-Ari, 2002; Marty and Llano, 2005). In some invertebrate systems, however, depolarizing responses to GABA involve cationic conductances associated with receptors that are pharmacologically or structurally distinct from those in the major vertebrate GABA receptor families (Yarowsky and Carpenter, 1978; Norekian, 1999; Swensen et al., 2000; Gutovitz et al., 2001; Beg and Jorgensen, 2003; Goaillard and Marder, 2003).

In the stomatogastric nervous system (STNS) of the crab Cancer borealis, GABA has been shown to elicit a depolarizing and two separate hyperpolarizing responses in motor neurons of the stomatogastric ganglion (STG). Vertebrate GABA receptor agonists elicited GABA-like responses in some but not all of the neurons, while antagonists were largely ineffective in blocking GABAergic actions (Swensen et al., 2000). The putative source of GABA for STG targets is eight GABA-immunoreactive axons that project from central ganglia into the STG (Nusbaum and Marder, 1989; Coleman and Nusbaum, 1994; Blitz et al., 1999; Swensen et al., 2000). Four of these fibers project through the ganglion into the nerves that innervate the stomach musculature (Swensen et al., 2000), suggesting that muscles proximal to these nerves might also be targets of GABA released by these fibers.

Here we report that one of these nearby muscles does, in fact, respond to GABA, and that this action is excitatory. Nerve-evoked contractions of the muscle are enhanced by micromolar concentrations of GABA, and the potentiation results, at least in part, from actions at the neuromuscular junction. Using a combination of immunohistochemical and biochemical approaches, we have also identified the most likely source of the GABA that modulates this muscle. We conclude that this GABA does not originate in the nearby nerves, but probably is produced and released by a distant neurohemal organ.

Materials and Methods

Animals and solutions

Jonah crabs Cancer borealis Stimpson were obtained from the Marine Biological Laboratory (Woods Hole, MA) and from Ocean Resources, Inc. (Sedgwick, ME) and were maintained in aerated seawater aquaria at 10 [degrees]C until used. Animals were anesthetized in ice for 30 min before all experiments. Physiological saline had the following composition: 440 mmol [1.sup.-1] NaCl, 11 mmol [1.sup.-1] KC1, 13 mmol [1.sup.-1] CaC [l sub.2], 26 mmol [1.sup.-1] MgC [l.sub.2], 5 mmol [1.sup.-1] maleic acid, 11 mmol [1.sup.-1] Trizma base, pH 7.4-7.5. The neurotransmitter [gamma]-aminobutyric acid (GABA), the GAB[A.sub.A] receptor agonist muscimol, the GAB[A.sub.A] receptor antagonist picrotoxin, and the GAB[A.sub.B] receptor agonist baclofen were purchased from Sigma-Aldrich (St. Louis, MO).

Contraction, excitatory junctional potential, and input resistance measurements

The nerve-evoked contractions, excitatory junctional potentials (EJPs), and input resistance were measured using the gastric mill 4 (gm4) muscle (nomenclature of Maynard and Dando, 1974), one of the largest muscles in the stomatogastric system. The basic procedures for these measurements have been previously described (Messinger et al., 2005; Le et al., 2006). Neuromuscular preparations consisting of the gm4 muscle and the dorsal gastric nerve (dgn), cut just below the stomatogastric ganglion (STG) were dissected from the stomach and placed in 5-ml petri dishes lined with Sylgard 182 (Dow Corning, Midland, MI). During recording sessions, the preparation was continuously superfused (4-5 ml/min) with saline that was cooled with an ice bath and regulated to within half of a degree at a temperature between 9 and 11 [degrees]C. Solutions containing GABA, or receptor agonists/antagonists, or both were bath-applied by means of a switching port at the inflow of the superfusion system.

For contraction measurements, one insertion of the muscle was pinned down to the dish, while the other was tied to an FT03 force displacement transducer (Astro-Med, West Warwick, RI) with a piece ([approximately equal to]3 cm) of size 6/0 silk suture thread (Fine Science Tools, Foster City, CA). The nerve was stimulated extracellularly with a train of 1-ms unipolar pulses produced by an isolated pulse stimulator (Model 2100, A-M Systems, Carlsborg, WA). This resulted in increased muscle tension, which was measured by the transducer. The transducer signal was amplified (Model 440, Brownlee Precision, San Jose, CA) by a factor of 10,000 and recorded using a Digidata 1322A acquisition system (Molecular Devices, Sunnyvale, CA).

For EJP and input resistance measurements, both insertions of the muscle were pinned to the dish. The dgn was stimulated with single pulses, and the resulting EJPs were measured with an Axoclamp 2B intracellular amplifier (Molecular Devices) using conventional 2 mol [1.sup.-1] KAc-filled microelectrodes with resistances of 7-10 M [OMEGA]. The Axoclamp signal was amplified 10-fold using the Brownlee amplifier and recorded using the Digidata system. Input resistance was measured using the Axoclamp amplifier in discontinuous current clamp mode. Hyperpolarizing (5-s duration, -20 nA) current pulses were injected, and input resistance was calculated as the change in membrane potential divided by the injected current.

Immunohistochemistry, antibodies, and microscopy

Wholemount immunohistochemistry was performed using techniques modified from those of Swensen et al. (2000). The stomatogastric nervous system (STNS) was dissected from the animal in physiological saline, fixed for 2-3 h in a solution of 4% paraformaldehyde (Sigma) in phosphate-buffered saline (PBS) at pH 7.0, and rinsed three times and incubated for 4 h in PBS containing 0.3% Triton X-100 (PBT). Samples were incubated in a rabbit polyclonal anti-GABA antibody (catalog No. A2502, Sigma-Aldrich; 1:200 final (dilution) and a mouse monoclonal anti-synapsin antibody (catalog No. 3C11, Developmental Studies Hybridoma Bank, University of Iowa; 1:10 final dilution) in PBT + 10% heat-inactivated normal goat serum (PHT) for 72 h at 4 [degrees]C. Both these primary antibodies have previously been used successfully in studies of the C. borealis STNS (Swensen et al., 2000; Goaillard et al., 2004; Le et al., 2006).

After incubation with primary antibodies, tissues were rinsed five times over 5 h in PBT and then incubated for 18-24 h with a mixture of Alexa Fluor 488-coupled goat anti-rabbit IgG (catalog No. A-11008, Invitrogen, Carlsbad, CA; 1:300 final dilution) and Alexa Fluor 594-coupled donkey anti-mouse IgG (catalog No. A-21203, Invitrogen; 1:300 final dilution) in PHT. After secondary antibody incubation, each preparation was rinsed five times over 5 h in PBT and then mounted on a glass slide in media consisting of 2.5% DABCO in 50% glycerol/50% Tween-TBS pH 9 solution. Fluorescently labeled tissue was viewed using a 40X Plan-Neofluar objective on a Zeiss Axioskop epifluorescence microscope (Peabody, MA). Images were collected and analyzed using a combination of IPLab 3.7 (BD Biosciences, Rockville, MA) and Photoshop software suite 8.0 (Adobe Systems, San Jose, CA).

Liquid chromatography and mass spectrometry (LC/MS)

Crab hemolymph was analyzed using a procedure similar to that of Garduno et al. (2002). Hemolymph was extracted from an anesthetized crab by inserting a #22 needle about 2 cm into the pericardial cavity through the seam between the carapace and abdomen in the middle of the posterior edge of the animal. A 3-ml syringe was used to extract 1-3 ml of hemolymph from each crab. Hemolymph samples were treated to minimize potential matrix effects in LC-MS analysis. First, the sample was precipitated with 1 mol [1.sup.-1] HCl (1:1, v/v) to remove proteins, and the resulting mixture was vortexed and filtered with 0.22-[micro]m cellulose acetate Costar spin-X centrifuge tube filters (Sigma-Aldrich) by spinning at 5600 X g for 10 min. Filters were discarded and the supernatant was stored at 4 [degrees]C until analysis.

Analyses were conducted using an Agilent Technologies 6100 series Quadrupole LC/MS system (Santa Clara, CA). Separations in the LC/MS system were achieved with an Agilent Eclipse XDB-C18 column (particle size 5 [micro]m, 4.6 X 150 mm). A binary gradient elution was used. Mobile phase A consisted of nanopure water, and mobile phase B consisted of anhydrous methanol. The elution profile was as follows: 0 min 77% A, 23% B; 0.5 min 55% A, 45% B; 10.5 min 50% A, 50% B; 12.5 min 50% A, 50% B; 13.5 min 77% A, 23% B; program ended at 14 min; flow rate was 500 [micro]l/min. Mass spectra were collected using electrospray ionization in positive ion mode with a capillary voltage of 3 kV (scanning range 50-1000 amu). A calibration curve was obtained by measuring peak areas from single-ion chromatograms (104.1 amu [+ or -] instrumental resolution of 0.5 amu) of standard solutions that contained 0.2, 0.4, 0.6, 0.8, 1, 2, or 4 [micro]mol [1.sup.-1] GABA.


A one-way ANOVA was used to analyze and compare the effects of 1[0 sup.-6] mol [1.sup.-1] and 1[0 sup.-5] mol [1.sup.-1] GABA on gm4 peak contractile force. The Holm-Sidak method was then used to make pairwise comparisons between the two concentrations and the control. In experiments in which only a single concentration of GABA or an agonist/antag- onist was tested, a paired Student's t test was used to determine statistical significance. All statistical tests were implemented using SigmaStat 3.1 (Systat Software, Point Richmond, CA). Statistical significance is indicated on figures using the following symbols: * P < 0.05, ** P < 0.01, *** P < 0.001. All uncertainties reported correspond to standard errors.

Figures were produced using the SigmaPlot 8.0 (Systat Software) and Canvas 9 (ACD Systems, Miami, FL) software packages.


Gamma-aminobutyric acid increases nerve-evoked contraction and the excitatory junctional potential of the gastric mill 4 muscle

Most stomatogastric muscles in Cancer borealis are innervated via one of the two lateral ventricular nerves (lvns) that branch from the dorsal ventricular nerve (dvn). The gastric mill 4 (gm4) muscle is one of the exceptions. The axon of the dorsal gastric motor neuron that innervates gm4 projects from the stomatogastric ganglion (STG) to the muscle through the dorsal gastric nerve (dgn) (Weimann et al., 1991) that splits from the dvn a few millimeters below the ganglion (Fig. 1). The precise location of the dvnllvn junction varies slightly from animal to animal but is typically a few millimeters above (i.e., dorsal to) the anterior portion of gm4. The dvn and the portions of the lvns that are proximal to gm4 contain GABA-immunoreactive fibers (Swensen et al., 2000). Although the lvns are not thought to innervate gm4, they are attached to gm4 muscle fibers by one or more small connectives, the function of which has never been determined.


Because of the proximity of gm4 to the GABAergic fibers in the lvns, we speculated that its contractions would be modulated in the presence of GABA. Neuromodulation of the C. borealis stomatogastric musculature has been extensively documented (Jorge-Rivera and Marder, 1996, 1997; Jorge-Rivera et al., 1998; Sharman et al., 2000; Verley et al., 2008). Nerve-evoked contractions of the gm4 muscle, in particular, can be modulated by the application of at least 10 substances that include small molecules (e.g., serotonin, dopamine) and peptides (Jorge-Rivera et al., 1998).

We tested the effect of two concentrations of GABA on gm4 contractions elicited by dgn stimulation (3-s duration, 12.5 Hz). The stimulation frequency was chosen to be comparable to that observed by Beenhakker and Nusbaum (2004) for dorsal gastric neuron spiking during gastric mill rhythms. The presence of 1[0.sup.-5] mol [1.sup.-1] GABA increased the peak gm4 contractile force, and this modulatory effect was reversible (Fig. 2A). Figure 2B summarizes the results of seven experiments done in 1[0 sup.-6] mol [1.sup.-1] GABA and seven experiments done in 1[0 sup.-5] mol [1.sup.-1] GABA. Contractile forces in GABA were measured about 15 min after beginning application, since the effect on contraction did not peak until more than 10 min after the initial exposure to GABA. Statistically significant differences in the mean peak contractile force for the control, 1[0 sup.-6] mol [1.sup.-1] GABA, and 1[0 sup.-5] mol [1.sup.-1] GABA data sets were found (one-way ANOVA, P = 0.002). The increase (65.3% [+ or -] 11.9% over basal) in the peak contractile force in the presence of 1[0 sup.-5] mol [1.sup.-1] GABA with respect to control was highly significant (Holm-Sidak method. P = 0.00066). With the application of 1[0 sup.-6] mol [1.sup.-1] GABA, the average peak force actually decreased slightly (1.9% [+ or -] 6.0%) with respect to control, but this effect was not significant (Holm-Sidak method, P = 0.86).


Gutovitz et al. (2001) previously found that GABA had both a presynaptic (increased transmitter release) and a postsynaptic (reduced muscle input resistance) action at the neuromuscular junction of several stomatogastric muscles in the lobster Homarus americanus. The presynaptic effect dominated the postsynaptic one, resulting in increased EJP amplitudes in GABA concentrations of 1[0 sup.-5] mol [1.sup.-1] and above. To determine whether similar mechanisms contribute to the increase in gm4 contraction amplitude in C. borealis, we measured the effect of GABA on unitary EJPs recorded from gm4b fibers. EJP amplitude increased in the presence of 1[0 sup.-5] mol [1.sup.-1] GABA (Fig. 3A). In Figure 3B, a summary of five such experiments, we see that average EJP amplitude increased by 27.7% [+ or -] 9.1% in the presence of GABA at this concentration and that the effect was statistically significant (paired t test, P = 0.033). When we measured the input resistance of gm4b muscle fibers, we found that the presence of 1[0 sup.-5] mol [1.sup.-1] GABA significantly reduced the average fiber resistance from 330 [+ or -] 54 k [OMEGA] (control) to 300 [+ or -] 54 k[OMEGA] (paired t test, n = 5, P = 0.012, data not shown), suggesting that the observed increase in gm4b EJP amplitude likely resulted from a presynaptic action of GABA.


Efforts to characterize the receptor responsible for modulation of gastric mill 4 by GABA

In the lobster study (Gutovitz et al., 2001), the vertebrate GAB[A.sub.B] agonist baclofen mimicked the GABA-evoked enhancement of EJPs. In C. borealis, Swensen et al. (2000) identified three distinct GABAergic responses in STG motor neurons and observed the vertebrate GAB[A.sub.A] agonist muscimol to preferentially activate an inward current, while baclofen had a weak or variable response. We studied nerve-evoked gm4 contractions in the presence of these substances in an effort to characterize the receptor responsible for the enhanced gm4 contraction. We used an electrical stimulation (3 s, 12.5 Hz) identical to that for the GABA contraction experiments shown in Figure 2 and applied agonists at the concentrations used by Gutovitz et al. (2001). The presence of muscimol (1[0.sup.-4] mol [1.sup.-1]) resulted in a small (9.0% [+ or -] 5.3%) statistically significant decrease, rather than an increase, in average peak contractile force (paired t test, n = 5, P = 0.019, data not shown). It was thus not surprising that the GAB[A.sub.A] antagonist picrotoxin did not block the GABA enhancement of contraction. Adding 1 [0.sup.-5] mol [l.sup.-1] GABA to a solution containing 1 [0.sup.-4] mol [1.sub.-1] picrotoxin increased the average peak contractile force by 44.5% [+ or -1] 11.4% (paired t test, n = 4. P = 0.052, data not shown). Application of baclofen (1 [0.sup.-4] mol [1.sup.-1]) increased peak force by 5.5% [+ or -1] 2.3%, but the effect was not quite statistically significant (paired t test, n = 5, P = 0.089, data not shown). The largest effect measured in the five baclofen experiments was an increase of 11.6%: in peak force.

Anatomical evidence for GABA release in the lateral and dorsal ventricular nerves

The dgn, which contains the motor axon that innervates gm4, does not contain GABA (Swensen et al, 2000). In the nearby dvn, however, four nerve fibers exhibit GABA-immunoreactivity, with two fibers projecting into each lvn (Swensen et al, 2000). The GABA-labeling in each lvn has been shown to be confined to the part of the nerve proximal to gm4 (Swensen et al., 2000), the portion where axons in the lvn make no known connections to muscles. The lvns themselves (and sometimes the dvn) do make small connections in this region onto gm4 muscle fibers (Fig. 1), and we postulated that these small nerves might contain branches of the GABAergic fibers found in the lvn.

To test this theory, we immunolabeled STNS preparations with antibodies against GABA and against the vesicle-associated protein synapsin. Both antibodies have been utilized successfully in the C. borealis STNS (Swensen et al., 2000; Goaillard et al., 2004; Le et al., 2006). but never simultaneously. The anti-synapsin antibody [alpha]-SYNORF1 (Klagges et al, 1996) has been used in the past to label synaptic structures (Goaillard et al., 2004), and we previously used it to show that the lvns are immunoreactive (Le et al., 2006). We first repeated these single-labeling experiments to show that the fluorophores did not bleed through when viewed with alternative filters. We then double-labeled and examined five STNS preparations, using staining of the STG and commissural ganglia to verify that our protocols were working. In each preparation, we observed GABA-and synapsin-imnrunoreactivity colocalized in one or two fibers in the dvn (Fig. 4A, B) and each lvn (Fig. 4C, D), with the staining largely punctate. In no instance, however, did we observe labeling by either antibody of one of the small nerves that connects the lvn to gm4 muscle fibers.


Detection of GABA in the crab hemolymph

At least 10 peptides and amines modulate contractions of stomatogastric musculature in C. borealis (Jorge-Rivera et al., 1998). Since motor neurons in the adult crustacean STNS do not appear to contain these substances as cotransmitters, the assumption has been that neuromodulation of the musculature is due to the actions of neurohormones that circulate in the hemolymph (Jorge-Rivera et al., 1998). In decapod Crustacea, neuromodulators are released into the hemolymph from two major neurosecretory structures: the sinus glands located in the eyestalks, and the pericardial organs (POs) in the pericardial cavity that surrounds the heart (Alexandrowicz, 1953; Cooke and Sullivan, 1982; Li et al., 2003; Billimoria et al., 2005; Fu et al., 2005). Nerve trunks in the POs but not the sinus glands immunolabel for GABA (Christie et al., 1995), and we postulated that the POs could be the source of the GABA that potentiated gm4 contractions.

To investigate this possibility, we extracted hemolymph from 12 C. borealis pericardial cavities. Each hemolymph sample was diluted and treated with acid to precipitate and remove protein before analysis. Reversed-phase liquid chromatography/mass spectrometry (LC/MS) of the filtrates revealed a hemolymph component in each of the 12 samples that eluted at a time precisely matching that of GABA standards (Fig. 5A). Single-ion chromatograms for the monovalent cation of GABA (m/z = 104.1) were extracted from the LC/MS data and consistently yielded a primary peak at the retention time of a standard GABA solution (2.7 min). With the combination of equivalent retention times and masses, we have high confidence in assigning this peak to GABA. All 12 hemolymph samples yielded a similar profile, with a primary GABA peak and two smaller flanking peaks. Spiking hemolymph samples with known amounts of GABA yielded increased peak heights only for the central peak, thereby confirming the assignment of GABA to the central peak. The minor peaks have not been identified.


We quantified GABA concentration by comparing the areas of the purported GABA peaks in the single-ion chromatograms to those of GABA standards ranging from 0.2-4.0 [micro]mol [1.sup.-1] (Fig. 5B), correcting for the factor of 2 dilution performed in sample preparation. Resolution between the primary GABA peak and side peaks was sufficient to provide highly accurate quantification. We found that GABA concentrations in the original samples ranged between 1.28 and 22.4 [micro]mol [1.sup.-1] (Table 1). The latter value appeared to be an outlier, as it was 3 times larger than the next highest concentration measured. It is possible that this elevated concentration was related to physiological activity of this particular crab. Excluding this anomalous result, the average GABA content in the hemolymph was determined to be 3.30 [+ or -] 0.66 [micro]mol [1.sup.-1]. This is comparable to the threshold concentration required to potentiate gm4 contractions, which we measured to be between [10.sup.-6] and [10.sup.-5] mol [1.sup.-1].


Gamma-aminobutyric acid as an excitatory neuromodulator of muscle contractions

While certain neuroactive substances are often thought of as "excitatory" or "inhibitory," it is not the molecule itself that determines the polarity of the action, and novel receptor types or atypical ion concentrations can result in unusual modulatory effects. GABA has previously been shown to potentiate muscle contractions in a handful of invertebrate and vertebrate systems (Devlin, 2001). Muscle contractions in response to GABA have been reported in the tube feet of several species of sea urchins (Florey et al., 1975) and starfish (Protas and Muske, 1980), the protractor muscle of sea cucumbers (Kobzar, 1984), and the striated muscle of the vas deferens of crayfish (Murdock, 1971). Distinct GABA receptors are responsible for the excitatory effects observed in these systems. For example, the crayfish receptors are indistinguishable from typical inhibitory receptors, in that GABA induces an increased permeability to C[l.sup.-]. The excitatory response is due to the unusual C[l.sup.-] concentrations in this system rather than to activation of an exotic receptor (Florey and Murdock, 1974). The sea urchin receptors have similar pharmacology to GAB[A.sub.A] receptors; however, their GABA response involves an enhanced permeability to N[a.sup.+] (Florey et al., 1975). GABA has also been shown to increase the resting tension of smooth muscle from the guinea pig (Kleinrok and Kilbinger, 1983; Taniyama et al., 1988). Even in this vertebrate system, two types of GABA receptors have been identified: (1) a "classical" receptor that also responds to muscimol and potentiates muscle contraction by increasing acetylcholine release and (2) an inhibitory receptor that is unaffected by typical agonists or antagonists.

Here we report for the first time that GABA can also enhance contractions in the crustacean stomatogastric nervous system (STNS). We also found that GABA increased the gastric mill 4 (gm4) muscle excitatory junctional potential (EJP) while decreasing muscle fiber input resistance, indicating that the effect on contraction is mediated at least partially by presynaptic actions at the neuromuscular junction. Excitatory GABAergic actions at the neuromuscular junction have been reported previously in the STNS. Gutovitz et al. (2001) observed that GABA increased EJPs and excitatory junctional currents in stomatogastric muscles in the lobster Homarus americanus, with a threshold concentration between [10.sup.-6] and [10.sup.-5] mol [1.sup.-1]. The effect of GABA in that system took 10-15 min to develop and was mimicked by application of the vertebrate GAB[A.sub.B] agonist baclofen, but it could not be blocked by any of the antagonists tested. The threshold concentration and time course for GABAergic enhancement of crab gm4 contractions were similar to what had been seen at the lobster neuromuscular junction, and so we predicted (incorrectly) that baclofen would similarly potentiate gm4 contractions. As in the crab stomatogastric ganglion (STG) (Swensen et al, 2000), we found that baclofen had a weak, variable effect on muscle contraction. Whether the GABA receptors in the crab and lobster musculature are from completely different families or differ only by a few amino acids (Wang et al, 1995; Mukherjee et al., 2006) remains an open question. We speculate that the latter is closer to the truth and that a metabotropic receptor that bears at least some structural similarity to a vertebrate GAB[A.sub.B] receptor is responsible for the enhancement of gm4 muscle contractions.
Table 1

Measured concentrations of [gamma]-aminobutyric acid (GABA) in Cancer
borealis hemolymph samples

Sample #   Peak area  [GABA] ([mu]mol [l.sup.-1])

1             122163                         6.97
2              78190                         4.46
3              87731                         5.00
4              26593                         1.52
5              22370                         1.28
6              25683                         1.46
7              22851                         1.30
8              37241                         2.12
9              25526                         1.46
10             77020                         4.39
11 *          393228                        22.42
12            112165                         6.40
Average *                                    3.30
S.E. *                                       0.66

Peak areas were determined from reversed-phase liquid
chromaatography-mass spectrometry single-ion chromalograms
(m/z = 104.1). GABA concentrations were calculated by comparing peak
areas to a calibration of standards (Fig. 5B) and correcting for sample

* Sample 11 was an outlier and not included in the calculation of
average GABA concentration.

GABA as a neurohormone

Due to the lack of anatomical evidence for nearby release, the 10 substances that have previously been shown to modify gm4 contractions (Jorge-Rivera et al., 1998) are presumed to be released outside the STNS, reaching the muscle as neurohormones. For exogenous application of each of these substances, the threshold concentration for a modulatory effect was less than [10.sup.-6] mol [1.sup.-1], and in most cases was below [10.sup.-6] mol [1.sup.-1] (Jorge-Rivera and Marder, 1996; Jorge-Rivera and Marder, 1997; Jorge-Rivera et al, 1998). For GABAergic modulation of gm4, we measured a threshold concentration ([10.sup.-6]-[10.sup.-5] mol [1.sup.-1]) that was at least an order of magnitude larger than that for the other modulators. The threshold concentration we measured was, however, comparable to that for GABAergic effects on lobster muscles (Gutovitz et al, 2001) and considerably smaller than the GABA concentrations used ([greater than or equal to][10.sup.-4] mol [1.sup.-1]) to induce changes in the activity of STG pyloric neurons in the lobster Homarus gammarus (Cazalets et al., 1987).

In our investigation we characterized two sources of GABA in the Cancer borealis STNS. First, we found GABA to be colocalized with synapsin in fibers in the dorsal ventricular nerve (dvn) and the lateral ventricular nerves (lvns), and hence it is probably released within those nerves. Four of the eight GABAergic fibers that enter the STG (Fig. 1) via the stomatogastric nerve (stn) are axons of the modulatory proctolin neurons (MPNs) (Swensen et al., 2000), and at least two MPN axons make it as far as the fans (M. Nusbaum, Univ. of Pennsylvania, unpubl. physiological obs.). Two more of the eight are axons of modulatory commissural neuron 1 (Swensen et al., 2000), but these axons do not project beyond the STG (Nusbaum et al., 1992). Four GABA-immunoreactive libers (at least two MPN axons and two others) exit the STG via the dvn (Swensen et al, 2000), and four proctolin-labeling fibers have also been found in the dvn (Marder et al., 1986). Thus it seems likely that MPN axons account for all of the GABA-labeling fibers in the dvn and lvns, although it is possible that two GABA-and two proctolin-staining fibers correspond to unidentified neurons.

With no labeling of the small connectives between the lvns and gm4, it is not clear how GABA released by MPN would make its way to the muscle. Moreover, it seems unlikely that GABAergic (or any other) modulation of gm4 by MPN would be physiologically relevant, since gm4 contracts during gastric mill rhythms, which themselves are suppressed by MPN activity (Blitz and Nusbaum, 1997, 1999). What seems more probable is that the GABAergic axons in the dvn and lvns make en passant synapses onto other axons in those nerves. In several STNS studies it has been demonstrated that local application of a neuromodulator to an axon can modify the spike trains that are transmitted to synaptic targets (Meyrand et al., 1992; Bucher et al., 2003; Goaillard and Marder, 2003). Axonal GABA receptors have not as yet been identified in the STNS, but they have been found in the vertebrate central nervous system (Charara et al., 2000; Kulik et al., 2003; Verdier et al., 2003; Trigo et al, 2007, 2008).

Our detection of GABA in the hemolymph extracted from the pericardial cavity points to the pericardia] organs (POs) as being a second source of GABA that could potentially modulate targets in the STNS. The POs have previously been shown to contain GABA (Christie et al., 1995), and until now it was assumed that this GABA modulated peptide release by the POs themselves and had no hormonal actions, since no distant target had been identified (Christie et al., 1995). Because the GABA concentrations that we measured in the hemolymph were close to the threshold for an effect on gm4 contraction, we conclude that the POs are the most likely source of the GABA that modulates the muscle. The notion of GABA as a crustacean neurohormone is not a new one: Kravitz et al. (1980) speculated this to be the case in H. americanus on the basis of observations of GABAergic effects on lobster muscle fibers and the lack of evidence for nearby release. In C. borealis, the STG and portions of connecting nerves are located within the anterior aorta, one of the main arteries connecting to the heart (Maynard and Dando, 1974; Steinacker, 1978), and injection of neuroactive substances into the pericardial cavity, which is several centimeters from the stomach, has been shown to modify the activity of gastric mill neurons in lobsters (Heinzel, 1988; Turrigiano and Selverston, 1990). Hemolymph pumped by the heart would reach muscles of the foregut not through the anterior aorta, but rather via branches of anterolateral arteries that also originate on the anterior side of the heart (Maynard, 1960). In the future it would be interesting to more precisely measure the GABA concentration and its temporal fluctuations in the immediate vicinity of the musculature, perhaps employing a microdialysis probe (Behrens et al., 2008). The crabs used in our study were starved for over a week. Levels of the peptide hormone cholecystokinin in the hemolymph of the lobster Panulirus interruptus were observed to increase about 400% above baseline in the hour after feeding (Turrigiano and Selverston, 1990). We speculate that GABA levels in C. borealis might also dramatically increase after feeding, reaching levels at which muscle contractions and foregut function could be modified appreciably.


We are indebted to B. Beltz, C. Billimoria, E. Marder, I. McGaw, D. Messinger, M. Nusbaum, and S. Pulver for valuable conversations and communications. We are grateful to M. Nusbaum, in particular, for sharing unpublished observations. We thank M. Lucas and A. Meyer for assistance in the laboratory. J. T. Birmingham acknowledges support from the Research Corporation, the Grass Foundation, and an award to Santa Clara University (SCU) under the Undergraduate Biological Sciences Education Program of the Howard Hughes Medical Institute. J. T. Birmingham and S. W. Suljak both acknowledge support from SCU. A. M. Lewis was supported by a DeNardo Science Scholar Award. The [alpha]-SYNORF1 antibody developed by E. Buch-ner was obtained from the Developmental Studies Hybrid-oma Bank developed under the auspices of the NTCHD and maintained by The University of Iowa, Department of Biology, Iowa City, TA 52242.

Literature Cited

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Received 6 August 2009; accepted 27 April 2010.

* To whom correspondence should be addressed. E-mail:

[dagger]Present address: Neuroscience Graduate Interdepartmental Program, UCLA. Los Angeles, CA 90095.

Abbreviations: dgn, dorsal gastric nerve; dvn, dorsal ventricular nerve; EJP, excitatory junctional potential; GABA, [gamma]-aminobutyric acid; gm4, gastric mill 4 muscle; lvn, lateral ventricular nerve; LC/MS, reversed-phase liquid chromatography-mass spectrometry; MPN, modulatory proctolin neuron; POs, pericardial organs; STG. stomatogastric ganglion; stn, stomatogastric nerve; STNS, stomatogastric nervous system.


(1) Departments of Chemistry and Biochemistry, (2) Biology, and (3) Physics; Santa Clara University, Santa Clara, California 95053
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Author:Suljak, Steven W.; Rose, Christopher M.; Sabatier, Christelle; Le, Thuc; Trieu, Quoc; Verley, Derek
Publication:The Biological Bulletin
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Date:Jun 1, 2010
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