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
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. Kleckner1,*
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
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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.
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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).
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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).
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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.
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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.
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© The Company of Biologists Ltd 2008
