Abstract:
Nitric oxide (NO) is an unconventional neurotransmitter. It is not stored in
vesicles and released by exocytosis. Rather, it is produced from arginine by the
activation of a calcium/calmodulin-dependent enzyme, nitric oxide synthase (NOS). NO
acts as an orthograde neurotransmitter in the central nervous system, which means that it
is released by pre-synaptic cells and affects post-synaptic cells. This unusual molecule
stimulates soluble guanylyl cyclase and increases intracellular levels of cGMP in the
post-synaptic cell. I wondered if an exogenous NO donor like sodium nitroprusside
would result in post-synaptic membrane depolarization and increased input resistance in
cells thought to receive this type of input. Using microelectrode techniques, I measured
resting potentials and action potentials in the MCC (metacerebral cell) in the cerebral
ganglion of Aplysia californica. I found that sodium nitroprusside caused a subthreshold
membrane depolarization and increase in input resistance. The literature suggests that
these effects may be the result of NÖ donors acting on potassium channels in the post-
synaptic membrane. These experiments support the idea that NO acts as a transmitter or
as a neuromodulator to influence the excitability of post-synaptic neurons in Aplysia.
Introduction
Nitric oxide (NO) is an unconventional neurotransmitter because it is not stored in
vesicles and released by exocytosis. Rather, it is produced from arginine by the
activation of the calcium/calmodulin-dependent enzyme, nitric oxide synthase (NOS).
NO acts as an orthograde neurotransmitter in the central nervous system by stimulating
guanylyl cyclase and increasing intracellular cGMP in the post-synaptic cell. The cGMP
increase stimulated by NO appears to alter one or more of the membrane ionic currents
that are required for spike/burst activity (Koh and Jacklet 2001). Several experiments
indicate that the potassium current may be strongly affected by NO.
Materials and methods
Animals and chemicals. Aplysia californica (100-150 g) were supplied by a company in
Florida that is associated with Eric Kandel's lab. The Aplysia were kept in an aquarium
tank at 16-20 degrees Celsius, and fed fresh seaweed from Agassiz Beach. Sodium
nitroprusside (420-003-M005, Alexis Corporation, San Diego, CA) solution (1 mM) was
made by dissolving .0002 g sodium nitroprusside in 1 mL of fresh seawater. The solution
was used immediately after mixing.
Electrophysiology. Isolated cerebral ganglia were incubated in fresh seawater before
desheathing at room temperature. A desheathed ganglion was pinned down on a sylgard
dish (1 inch in diameter) and superfused with seawater to measure the subthreshold
membrane depolarization and increase in input resistance of the MCC in response to
sodium nitroprusside. Intracellular recording electrodes were filled with 3 M KCl.
Membrane depolarizations were recorded immediately after the end of the sodium
nitroprusside superfusion. Input resistance was measured as the voltage deflection caused
by a current pulse. The EPSP in the MCC was evoked by stimulating the cell with a 2
second current pulse injection, resulting in a train of action potential spikes. The firing
frequency of MCC during stimulation was moderately stable most of the time. Because
of the tendency to facilitate, the EPSPs were measured with at least 15 sec breaks for
recovery to the normal state before subsequent stimulation.
Figure 1. Dorsal (A) and ventral (B) views of the cerebral ganglion in Aplysia californica.
From Joyce K. Ono.
Results
Initially, T had hoped to make pre-synaptic intracellular recordings from C2, a
small cell in the cerebral ganglion, and post-synaptic intracellular recordings from MCC.
the metacerebral cell, its follower in the same ganglion. However, the position of C2
within different specimens of Aplysia is extremely variable, and identification of the
correct cell was difficult. I shifted gears, and looked to see if NO released from an
exogenous NO-donor compound like sodium nitroprusside would mimic the membrane
depolarization and increase in input resistance that are characteristic of the EPSP that
occurs in the MCC during stimulation of NOS-containing neuron C2 (Jacklet 1999).
Sodium nitroprusside induced membrane depolarization and an increase in input
resistance.
Action potentials were fired about once every two seconds in the MCC before
application of sodium nitroprusside. Depolarizing current applied for 2 seconds resulted
in a train of action potentials, separated by 50 ms. Immediately following the NO donor
superfusion, the same amount of depolarizing current was applied and EPSPs. I interpret
this as an increase in input resistance. The cell recovered after sodium nitroprusside
application, firing more than once a second on average.
Figure 2. A graph of firing frequency vs. time. Sodium nitroprusside was applied at
200 ms.
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1.4

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0.6

.
0.2
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500
1000
1500
2000
Discussion
The time scale for nitric oxide’s role in synaptic plasticity is incredibly short. NO
is highly reactive and extremely labile, so its biological half-life is within a range of only
a few seconds, and it is oxidized to stable nitrite and nitrate (Rode 1992). The story of
NO in synaptic transmission is therefore fast-paced. NÖS enzymes can be discriminated
as inducible or constitutive. The brain and endothelial forms are constitutive in that
stimuli for NO formation do not typically result in new enzyme protein synthesis.
Instead, in the brain a stimulus (such as glutamate) acting at NMDA receptors triggers
calcium. Calcium binds to calmodulin, thereby activating NOS. This mode of activation
explains the ability of glutamate neurotransmission to stimulate NO formation in a matter
of seconds.
The discovery of nitric oxide as a neurotransmitter has radically altered our
thinking about synaptic transmission. Being a labile, free radical gas (though in most
biological situations NO is in solution), NO is not stored in synaptic vesicles. Instead it is
synthesized as needed by NO synthase (NOS) from its precursor L-arginine. Rather than
exocytosis, NO simply diffuses from nerve terminals. It does not react with receptors but
diffuses into adjacent cells. In place of reversible interactions with targets, NO forms
covalent linkages to a multiplicity of targets that may be enzymes, such as guanylyl
cyclase (GC) or other protein or nonprotein targets. Inactivation of NO presumably
involves diffusion away from targets as well as covalent linkages to an assortment of
small or large molecules such as superoxide and diverse proteins (Snyder 2001).
Soluble GC is known as a major target protein for NO in the central nervous
system. NO activates sGC and promotes the increase in intracellular cGMP level by
binding to the heme moiety with high affinity. Soluble GC and cGMP were thought to be
involved in the second messenger pathway for NO action in the MCC because a previous
study showed that the EPSP was partially suppressed by methylene blue, a nonspecific
GC inhibitor (Jacklet and Gruhn 1994). The depolarization resulting from sodium
nitroprusside treatment is consistent with the idea that they cause closure of potassium
channels, and the membrane potential shifts away from the potassium equilibrium
potential, in the depolarizing direction, as a result. The depolarization and decrease in
membrane conductance suggest that potassium channels are closed. NO depolarizes the
MCC by reducing a resting membrane potassium conductance, and this effect is
mimicked by sodium nitroprusside.
These results seem contradictory because an increase in depolarization should not
theoretically be found with an increase in input resistance, but NO seems to have a
variety of actions on membrane ion channels. Neurotransmission in general is surely
many times more complex than we have the understanding for right now.
Conclusion
Numerous mammalian studies strongly implicate NO in learning, especially LTP
and LTD. A recent study provides strong evidence of NO's involvement in the formation
of olfactory memories in sheep (Kendrick et al. 1997) and provides a link to the
conserved olfactory function for NÖ signaling. Ewes learn to recognize the odor of their
lambs within 2 hrs of giving birth. NOS inhibitors block this learning and infusion of NO
by microdialysis reverses the block.
Literature cited
Jacklet, J.W. and M. Gruhn. 1994. Nitric oxide as a putative transmitter in Aplysia: neural
circuits and membrane effects. Neth J Zool 44: 524-534.
Kendrick, K., Guevara-Guzzman, R., Zorrilla, J., Hinton, M., Broad, K., Mimmiack, M.
and S. Öhkura. 1997. Formation of olfactory memories mediated by nitric oxide.
Nature. 388: 670-674.
Koh, H.-Y. and J.W.Jacklet. 2001. Nitric oxide induces cGMP immunoreactivity and
modulates membrane conductance in identified central neurons of Aplysia.
European Journal of Neuroscience. 13: 553-560.
Kupfermann, I. and K.R. Weiss. 1982. Activity of an identified serotonergic neuron in
free moving Aplysia correlates with behavioral arousal. Brain Res 241: 334-337.
Rode, B. 1992. Nitric oxide as a messenger molecule and its clinical significance. Neuron
8: 3-11.
Weiss, K.R., Shapiro, E. and I. Kupfermann. 1986. Modulatory synaptic actions of an
identified histaminergic neuron on the serotonergic metacerebral cell of Aplysia. J
Neurosci 6: 2393-2402.