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First published online February 15, 2008
Journal of Experimental Biology 211, 824-833 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.011866

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Pharmacology of ionotropic and metabotropic glutamate receptors on neurons involved in feeding behavior in the pond snail, Helisoma trivolvis

Elizabeth Scannell¹, Carly A. Dell'Ova¹, Elizabeth M. Quinlan², A. Don Murphy³ and Nancy W. Kleckner^1,*

¹ Program in Neuroscience, Bates College, Lewiston, ME 04240, USA
² Department of Biology, Neuroscience and Cognitive Sciences Program, University of Maryland, College Park, MD 20742, USA
³ Department of Biological Sciences and Laboratory of Integrative Neuroscience, University of Illinois at Chicago, Chicago, IL 60607, USA

View larger version (36K): [in this window] [in a new window]   Fig. 1. Helisoma buccal neuron B5 is inhibited by glutamate and low concentrations of kainite (KA). (A) Diagram of the buccal ganglia showing the bilateral location of left and right (shaded in grey) B5 neuron cell bodies (adapted from Quinlan et al., 1995). (B) A right B5 neuron was injected with Lucifer Yellow. Its main axon extends out through the ipsilateral esophageal trunk. Fine processes extending from the cell body are occluded by the brightness of the cell body staining. Fluorescence image is layered on a DIC image of the same field (see Materials and methods). Scale bar, 100 µm. (C) Glutamate (Glu) dose-dependently reduced action potential (AP) frequency in a B5 neuron. Membrane potential was measured using standard intracellular recording techniques. Glutamate was perfused during the times indicated by the horizontal bars. Arrowhead indicates 0 mV. (D) Dose-dependent reduction by glutamate of mean (±s.e.m.) AP frequency (*P<0.05, N=5). (E) KA (10 µmol l–1) appeared to reduce the AP frequency in a B5 neuron. The effect was blocked by 50 µmol l–1 CNQX. Drugs were perfused during the times indicated by the horizontal bars. Arrowhead indicates 0 mV. (F) Mean (±s.e.m.) AP frequency of B5 neurons (N=9) during perfusion of KA (10 and 30 µmol l–1) and/or 50 µmol l–1 CNQX. ANOVA indicated a significant effect of 10 µmol l–1 KA, 50 µmol l–1 CNQX and the combined KA/CNQX. The increase in AP frequency with 30 µmol l–1 KA was not significant, whereas the decrease in frequency with 10 µmol l–1 KA was significantly different from control (*P<0.05, t-test, N=9). ET, esophageal trunk; LBN, lateral buccal nerve; VBN, ventral buccal nerve; CBC, cerebrobuccal connective.

View larger version (38K): [in this window] [in a new window]   Fig. 2. Helisoma buccal neuron B19 is inhibited by glutamate and excited by KA. (A) Diagram of the buccal ganglia showing the bilateral location of left and right (shaded in grey) B19 neuron cell bodies (adapted from Quinlan et al., 1995). (B) A right B19 neuron was injected with Lucifer Yellow. Its main projections extend ipsilaterally out through the right VBN and LBN, and contralaterally out through the left VBN. Extensive neurites leave the cell body and axonal projections; some are obscured by the bright cell body staining. Fluorescence image is a composite of 31 separate images and is layered on an HMC image of the same field (see Materials and methods). Scale bar, 100 µm. (C) Glutamate, applied for the durations indicated by horizontal bars, dose-dependently reduced AP frequency in a B19 neuron. Breaks in the trace represent 2–3 min of saline perfusion. Arrowhead indicates 0 mV. (D) Glutamate dose-dependently reduced mean (±s.e.m.) AP frequency of B19 neurons (*P<0.05, N=9). (E) KA had no effect on mean (±s.e.m.) AP frequency at 10 µmol l–1 (N=9), but significantly increased frequency at 30 µmol l–1 (+P<0.03; N=13). (F) CNQX had no effect on mean (±s.e.m.) AP frequency in B19 neurons (N=9).

View larger version (47K): [in this window] [in a new window]   Fig. 3. Helisoma buccal neuron B27 is inhibited by glutamate and excited by KA. (A) Diagram of the buccal ganglia showing the bilateral location of left and right (shaded in grey) B27 neuron cell bodies (adapted from Quinlan et al., 1995). (B) A left B27 neuron was injected with Lucifer Yellow. Its main axons extend out through the ipsilateral and contralateral LBN. Fine processes extending from the cell body and axons are partially occluded by the brightness of the cell body staining. The fluorescence image is a composite of 17 separate images and is layered on a DIC image of the same field (see Materials and methods). Scale bar, 100 µm. (C) Glutamate dose-dependently reduced AP frequency in a B27 neuron. Membrane potential was measured using standard intracellular recording techniques. Drugs were perfused during the time indicated by the horizontal bars. Breaks in the recording reflect 1–2 min of saline perfusion. Arrowhead, 0 mV. (D) Dose-dependent reduction by glutamate of mean (±s.e.m.) AP frequency of eight cells (*P<0.01). (E) Mean (±s.e.m.) AP frequency of B27 neurons was increased during perfusion of 30 µmol l–1 KA (#P=0.0002, N=10), but not 10 µmol l–1 KA (N=9). (F) CNQX (50 µmol l–1) had no effect on KA-induced excitation of some B27 neurons (upper trace), and inhibited excitation in others (lower trace). Drugs were perfused during the time indicated by the horizontal lines. Arrowheads indicate 0 mV. (G) CNQX reduced the KA-induced increase in mean (±s.e.m.) AP frequency in B27 neurons (+P<0.05, N=9).

View larger version (8K): [in this window] [in a new window]   Fig. 4. KA-induced excitation of neuron B19 is due to electrotonic coupling with neuron B27. (A) Electrotonic coupling between neurons B19 and B27 in the buccal ganglia of Helisoma is shown during simultaneous intracellular recordings of neurons B19 and B27. DC hyperpolarization of neuron B19 produced a hyperpolarization in the membrane potential of neuron B27. A coupling coefficient could not be quantified due to the presence of a slight bridge imbalance. (B) Application of 10 µmol l–1 KA (arrowhead) had no apparent effect on the membrane potential of an isolated B19 neuron (top trace). In the same neuron, 1 mmol l–1 glutamate (arrowhead) produced a pronounced hyperpolarization of the membrane potential. Horizontal calibration bar indicates 10 s in the top trace and 20 s in the bottom trace. HS, Helisoma saline.

View larger version (36K): [in this window] [in a new window]   Fig. 5. Agonist pharmacology of inhibitory glutamate receptors in Helisoma buccal neurons. (A) ACPD, DCG-IV and quisqualate (Quis) reduced AP frequency in individual B5, B19 and B27 neurons, respectively. Membrane potential was measured with standard intracellular recording techniques. Drugs were applied during the times indicated by the horizontal lines. Arrowheads indicate 0 mV. (B) mGluR agonists Quis (10 µmol l–1) and ACPD (100 µmol l–1) significantly reduced mean (±s.e.m.) AP frequency of B5 neurons (*P<0.007, N=15 and 13, respectively). DHPG, DCG-IV and L-AP4 (all N=6) had no effect on firing rate. (C) Quis (10 µmol l–1) significantly reduced mean (±s.e.m.) AP frequency in B19 neurons (#P<0.0001, N=20). ACPD (N=14), DHPG (N=9), DCG-IV (N=8) and L-AP4 (N=7) had no effect on AP frequency. (D) Quis (10 µmol l–1) significantly reduced mean (±s.e.m.) AP frequency of B27 neurons (+P<0.05, N=9), whereas ACPD (N=4), DHPG (N=8), DCG-IV (N=8) and L-AP4 (N=7) had no effect.