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ABSTRACT AND INTRODUCTIONReduced glutathione, GSH, induces the signature feeding behavior in Hydra vulgaris (Loomis, 1955; Lenhoff, 1961). The behavior consists of mouth opening, tentacle writhing, and mouth closing. The duration of mouth opening is prolonged by the inhibitory neurotransmitter, gamma-amino-butyric acid, GABA (Pierobon, et al., 2001). It is possible to record the electrical effects of GSH, GABA, the GABAB agonist, baclofen, and the GABAB antagonist, phaclofen in detached hypostome preparations. The hypostome of Hydra vulgaris has two circumferential nerve rings; one that runs beneath and below the tentacles (proximal nerve ring) and one that runs midway between the mouth and the inter-tentacular ring (distal nerve ring) (Hufngael and Kass-Simon, 2015). A suction electrode placed on the apex of the hypostome covers the mouth, its surrounding sensory cells, and the distal nerve ring, but not the cells at the base of the hypostome, or the proximal, inter-tentacular nerve ring. In this way it is possible to record the differential effects of GSH and GABAB ligands at the mouth and the hypostomal dome.

GSH and Metabotropic GABABReceptor Ligands affect the Electrical Activity at Hydras Hypostome B.M. Lauro and G. Kass-Simon Interdisciplinary Neurosciences Program, University of Rhode Island, Kingston, RI USAFig. 4aMATERIALS AND METHODS

I. AnimalsHydra vulgaris, raised at 18 1.0C in BVC (Loomis and Lenhoff, 1956) were randomly selected, 24 2 hours after having been fed with brine shrimp ad liberatum. Hydra heads and tentacles were ablated from the body of the experimental animals; tentacles were cut below the tentacle insertion region; the excised heads were allowed to heal for 24 2 hours before electrical recordings.

Excised Hypostome with ablated tentacles

II. Recording MethodsElectrical recordings were conducted as previously described (Passano and McCullough, 1964, Kass-Simon, et al., 2003). Extracellular recordings were made with a suction electrode attached directly at the mouth opening of the hypostome. Impulses from the suction electrode were delivered to a differential amplifier, converted to digital output and visualized using LabChart 7 software (AD Instruments) on a computer. During recording, the preparations were observed through a dissecting microscope at 100X magnification.

Suction electrode recording from the apex of the hypostome. GSH, with or without ligands, is applied either to the bath with BVC in the pipette or to the pipette with BVC in the bath.

III. Ligands. The following ligands were used: reduced glutathione (GSH), GABA, and the GABAB agonist and antagonist, baclofen and phaclofen. Test substances were made fresh at 10-fold their final concentration and were subsequently diluted.

IV. Data AnalysisResults were analyzed using Friedmans Analysis of Variance (FANOVA) or Welchs T-Test. RESULTS I. GSH added to the bath increases small and medium sized impulses. Continuous recording in BVC (5 minutes) followed by a 10-minute recording period in GSH 5x10-6. Small pulses are indicated by diamonds and medium pulses are indicated by squares.

Control (BVC) GSH (5x10-6)

II. GSH at concentrations of 5x10-8M, 5x10-7M or 5x10-6M added to the bath reduces the number of bursts of pacemaker pulses. However, GSH added to the mouth only reduces pacemaker pulses at the highest concentration. Samples were taken from equivalent times after the addition of the test substance in each period.

Control (BVC)GSH (5x10-8)



III. However, when ligands are placed within the pipette, at the mouth (exposing the mouth, the oral sensory cells, the distal nerve ring, and oral endoderm), only the highest concentration of GSH (5x10-6) inhibits pacemaker bursts. Samples are taken from comparable recording periods.


IV. At the mouth, addition of GABA to GSH increases the number of small pulses and the number pacemaker bursts. These are sample recordings from comparable periods.

GSH 5x10-7 GSH 5x10-7 + GABA

V. Baclofen in the presence of GSH (5x10-7) at the mouth decreases small impulses. This effect was abolished by phaclofen.

GSH 5x10-7 GSH 5x10-7 + Baclofen

GSH 5x10-7GSH 5x10-7 + Phaclofen

Our experiments indicate that receptors for GSH and GABA exist at the mouth and at the dome of the hypostome.II. GSH differentially affects the electrical activity near the mouth and at the dome: At both dome and mouth, GSH decreases pacemaker bursts, although the decrease only occurs at the highest concentration at the mouth, suggesting that there may be more GSH receptors or receptor connections at the dome. GSH increases electrical activity the mouth and dome, but the electrical activity (at concentrations less than 5x10-7) at the mouth is greater than at the dome. GABA increases small and medium impulses and pacemaker bursts at the mouth. But the GABAB agonist, baclofen, decreases small impulses at the mouth, suggesting that the increase may be due to GABAA receptors. This is consistent with the prolonged mouth opening due to GABAA receptors (Pierobon, 2015). The GABAB antagonist, phaclofen, increases the number of bursts at the dome, suggesting a blockade of GABAB inhibition. In summary, our data suggests that GABA acting on metabotropic receptors modulates the electrical activity that causes the GSH-induced mouth opening response. SUMMARY

Literature citedGrosvenor, W. et al., 1996. Chemical Senses, 21: 313-321.Kass-Simon, G. et al., 2003. Comp. Biochem. Physiol. A, 136: 329-342.Kass-Simon, G, et al., 2007. Comp. Biochem. Physiol. A, 1446: 9-25. Kinnamon, J.C., and J.A. Westfall. 1981. J. Morphol., 168: 321-329. Lambert, M. et al., 2010. VDM Publishing. Loomis, W.F., and H.M. Lenhoff. 1956. J. Exp. Zoology, 132: 555-573. Pierobon, P. et al., 1995. Life Sci., 56: 1485-1497.Pierobon, P. 2015. J. Exp. Biology, 218: 2226-2232.

Printing service provided by the RI-INBRE Centralized Research Core Facility.

Acknowledgements

VI. The GABAB antagonist, phaclofen, increases the number of bursts at the dome relative to GSH, counteracting the decrease in bursts caused by GSH relative to BVC. This effect was abolished with baclofen.

GSH 5x10-7GSH 5x10-7 + Phaclofen

GSH 5x10-7GSH 5x10-7 + BaclofenVII. In general, the aggregate of all impulses caused by GSH or GABA ligands was significantly greater when applied directly to the mouth than around the dome.

GSH 5x10-7 (Dome)GSH 5x10-7 (Mouth)

GSH 5x10-7 + GABA (Dome)GSH 5x10-7 + GABA (Mouth)

GSH 5x10-7 + Baclofen (Dome)GSH 5x10-7 + Baclofen (Mouth)

GSH 5x10-7 + Phaclofen (Dome) GSH 5x10-7 + Phaclofen (Mouth)

RESULTS CONTD

A portion of this work was funded by the enhancement of Graduate Study Research Award received from the University of Rhode Island Graduate School. We thank the RI-INBRE Centralized Research Core Facility for the printing of this poster.

AmplifierPipette TipBVC/Ligand

24 +/- 2 hrs

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