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TINS November i 985
478
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Neural and molecular mech isms underlying
in rrnation storage in Aplysia: imp.cations for
learning and memory
John H. Byrne
The marine mollusc Aplysia /s one of several experimental preparations that
neuroscientists are using to help understand the mechanisms underlying learning
and memory. Simple behavioral modifications such as sensitization and classical
conditioning can be related to changes in the ability o f previously formed synaptic
connections to release neurotransmitter substance. The modifications of
transmitter release in turn are regulated by the intracellular second messengers
Ca2+ and cAMP. The second messenger systems may act separately in the case o f
non-associative learning (sensitization) but their specific interactions may underly
forms o f associative learning such as classical conditioning.
A fundamental problem in neurobiology is to understand the basic
mechanisms of learning and memory.
What parts of the nervous system are
involved in learning and memory, how
is information about a learned event
acquired and encoded at the cellular
level, how is the information stored,
and once stored how is it retrieved or
read-out? These are questions for
which few answers are available at a
mechanistic level. While most neuroscientists believe that changes in individual nerve cells (or at least small
groups of cells) are critical for learning
and memory, there is considerably less
agreement on the underlying mechanisms for such changes. Mechanisms
such as growth of new synaptic contacts, alterations in synaptic efficacy at
previously existing synapses, and reverberatory activity in neural circnits~
have been proposed, but only
recently have they been the subject of
direct experimental analyses. Considerable attention is now being focused on the hypothesis that a change
in synaptic transmission at previously
formed synaptic connections may
underlie examples of learning such as
habituation, sensitization and classical
conditioning. A number of experimental preparations are being examined to test this hypothesis and to
study the neural mechanisms of learnings . One that seems particularly
useful for a molecular analysis is the
marine mollusc Aplysia, the subject of
this review. This animal has a relatively simple nervous system with
large, identifiable neurons that are
accessible for detailed biochemical and
biophysical studies.
© 1985, Elsevier Science Publishers B.V., Amsterdam
The connections between sensory
neurons and motor neurons controlling the defensive siphon withdrawal
reflex and the defensive tail withdrawal reflex, have been the focus of
recent analyses6,7. These connections
exhibit a number of plastic properties
including synaptic depression, posttetanic potentiation, presynaptic facilitation,
activity-dependent neuromodulation and long-term potentiation6-1 a. The synaptic modifications in
turn have been related to a variety of
behavioral modifications including
habituation, sensitization and classical
conditioning7,9,N .
Neural and molecular mechanisms
contrilmting to ram-associative
information storage
Neural and molecular mechanisms
contributing to sensitization have been
analysed extensively in the siphon and
gill withdrawal reflex of Aplysia, and
similar mechanisms appear to contribute to sensitization of the tail withdrawal reflex as well. Sensitization is a
simple form of non-associative learning
in which a non-specific enhancement of
the response to a test stimulus is
produced by a second averswe stimulus applied to another part of the
animal. For example, a weak mechanical stimulus delivered to the siphon of
Aplysia produces a small withdrawal
of the siphon and nearby gill. If,
however, a noxious stimulus is applied
to the animal's head or tail. subsequent
test stimuli delivered to the siphon
produce much greater responses 7.
Both short-term sensitization, lasting
tens of minutes, and long-term sensitization, lasting days to weeks, have
been analysed in Aplysia but the
mechanisms for the short-term effects
are best understood and will be the
focus of this review.
Sensitizing stimuli to one part of the
animal lead to the release of a
modulatory transmitter that appears to
produce presynaptic facilitation of
many sensory neurons including those
that do not have their receptive fields
in the region of the sensitizing stimulus. The effects of presynaptic facili-
Sensitizing
stimulus
to animal
Activation of
modulatory
intemeurons
Modulatory,4,..--~-b ' ~ ' ~ " ~ - ' - ' ~ - - '~- ' ~ ~
transmitter
adenylate cyclase
release by
~'~activity in
intemeurons
~
Increased
levels
of
cyclic AMP
Increased ~ . . . . ~ - - " ~
activity of
protein kinase
Increas.ed
~ protein
phosphorylation
S. p i k ~ . . . . . . . . . . _ . ~ ~ a s e d
m ~
Ca2+ influx
sensory neurons
~ !nto
broadened
terminal
Increased.,4~
EPSP in
motor neurons
Increased
.~_~activation of
motor neurons
~
Decreased
,~_ K +
....-- conductance
Increased
transmitter
~ release from
....--- sensory neurons
Enhanced
. ~ behaviora!
response
Fig. 1. Sequenceof events contributing to sensitizationin Aplysia. Modifiedfrom Ref. 23.
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479
N o v e m b e r 1985
tation are expressed when subsequent
test stimuli are presented. Presynaptic
facilitation leads to an enhanced release of neurotransmitter from sensory
neurons and thus an enhanced probability of activating the motor neurons
and an enhanced behavioral response
(Fig. 1). The effects of the natural
neuromodulator released by sensitizing stimuli can be mimicked by
application of serotonin or SCP13
('small cardio-acceleratory peptide
B') 11-15. These chemicals, like the
natural modulator, exert their effects
through changes in the level of the
intracellular
second
messenger
cAMP 14-21. One consequence of the
elevated cAMP levels is reduction of a
resting steady-state K + current by the
closure of an 'S-channel' (so named
because it is serotonin-sensitive) that
contributes significantly to the repolarization of the action potential ~5.22. As
a result of closure of the S-channel,
repolarizing K + currents are decreased and action potentials initiated
in sensory neurons by test stimuli are
broader. Consequently, the Ca 2+ influx that normally occurs during the
action potential is prolonged. This
allows for greater transmitter release
and the enhanced activiation of the
motor neurons, that contributes to
sensitization.
Since short-term sensitization and
changes in synaptic efficacy that contribute to sensitization are relatively
persistent (15-30min), an obvious
question concerns which of the biochemical steps illustrated in Fig. 1 is
critical for storing the short-term
memory. To address this issue Castellucci et al. 23 injected an inhibitor of
cAMP dependent protein kinase into
sensory neurons just after the application of serotonin. As a result of the
injections the extent of the spike
broadening was decreased dramatically23. Thus, the duration of the
memory for the short-term sensitization seems to depend primarily on the
persistence of an active protein kinase
and not the persistence of a phosphorylated protein. What then determines the time-course of cAMP levels
that keep the protein kinase active?
Two possibilities must be considered.
One is the time-course of cAMP
degradation (controlled by phosphodiesterase) and the second is the
time-course of cAMP synthesis. To
examine the possible role of increased
synthesis, Castellucci et al. 24 utilized
GDPI~S, an irreversible inhibitor of the
cyclase. Injection of GDP[3S just after
the spike broadening induced by
serotonin caused a rapid return of the
spike to its pre-serotonin duration 24,25.
These results indicate that the elevation of cAMP levels is dependent upon
continued activation of the cyclase and
that a molecular locus for the shortterm memory is at the level of the
adenylate cyclase complex.
Neural model of associative learning
While the analysis of short-term
sensitization is fairly extensive 7, little
was known until recently about the
types of storage mechanisms that may
underly memories produced by associative learning. One hypothesis is that
the mechanism contributing to the
associative learning in this animal is
simply an elaboration of mechanisms
already in place that contribute to
sensitization.
Before discussing this possibility it is
useful to review briefly the features of
associative learning and compare them
with sensitization. One example of
associative learning that has been
examined extensively is classical or
Pavlovian conditioning. In classical
conditioning, a test stimulus (known as
the conditioned stimulus, CS) becomes effective in eliciting a response
(the conditioned response, CR) when
the CS is paired with another stimulus
(the reinforcing or unconditioned
stimulus, US) that reliably produces a
response (the unconditioned response,
UR). For example, in salivary conditioning the tone (CS) becomes effective in eliciting salivation (CR) if the
tone is paired repeatedly with another
stimulus (US, e.g. meat powder) that
reliably produces a response (UR).
Classical conditioning and sensitization share some common features. In
both, the effectiveness of a test
stimulus in eliciting a response is
modified by a reinforcing stimulus.
They differ, however, in that classical
conditioning requires a close temporal
association between the CS and the
reinforcer (US). If the pairing of the
tone and meat powder are separated in
time, the effectiveness of the tone in
eliciting salivation is reduced. Response enhancement that occurs independent of the timing of the CS and
US is sensitization.
At the mechanistic level how might
sensitization and classical conditioning
be related? One possibility is that
electrical activity in the CS pathway is
capable of amplifying the effects of
sensitizing or reinforcing stimuli on the
CS pathway. Such a notion is consistent with the theoretical work of some
neurobiologists and psychologists who
have assumed that the formation of
associations depends upon the contiguous activation of sensory pathways
and modulatory arousal centers~-~L
A general model of how sensitization and classical conditioning might
be related at the cellular level is
A. LEARNING
B. MEMORY
PAIRED
R
STIMULUS .-D,,.(
E
S
P
0
G
UNPAIRED
STIMULUS "~" I
-<•
O
I
~
s
E
SYNAPSE(OUTPUT)
OMA AND DENDRITES(INPUT)
PAIREDACTIVITY
Fig. 2. General model of activity-dependent neuromodulation. (A) Learning. Stippling indicates
temporally contiguous activity. A motivationally potent reinforcing stimulus activates a neural
response system and a modulatory system that regulates the efficacy of diffuse afferents to the response
system. Increased spike activity in the paired afferent (1) immediately before the modulatory signal
amplifies the degree and duration o f the modulatory effects, perhaps through the Ca2+ sensitivity of the
modulatory evoked second messenger. The unpaired afferent neuron (2) does not show an
amplification of the modulatory effects. (B) Memory. The amplified modulatory effects cause longterm increases in transmitter release and~or excitability of the paired neuron, which in turn strengthens
the functional connection between the paired neuron(l) and the response system. Modified from Ref. 8.
77NS November 1985
480
illustrated in Fig. 2. Assume that two
sensory pathways, designated as sensory neuron 1 and sensory neuron 2,
make weak subthreshold connections
to a response system. The two sensory
neurons might represent two cells of
the same modality with different
receptive fields or sensory neurons
representing two different modalities.
(In general the two input cells could be
interneurons as well.) Reinforcing
stimuli have two effects. One is to
activate directly the response system
and produce the unconditioned response (UR). The second is to activate
a diffuse modulatory or facilitatory
system that non-specifically enhances
the connections of all the sensory
neurons. Reinforcing stimuli may enhance synaptic transmission by initiating an increase in intracellular cAMP
levels, which in tum affect neurotransmitter release through the sequence of
events illustrated in Fig. l. The
properties of the system at this level
are rather non-specific since the modulator influences all of the target
sensory neurons and this modulation
occurs whether or not there is a CS.
How is the temporal specificity characteristic of associative learning obtained
from such a seemingly non-specific
system? At the cellular level, temporal
specificity could be achieved if input
from the CS can amplify the biochemical sequence of events initiated by the
reinforcing stimulus (or US). One
possibility is that spike activity in a
sensory neuron which occurs just prior
to the modulatory US causes a selective amplification of cAMP levels over
and above the increase caused by the
modulatory input alone. Thus, the
extent of the modulatory effects in a
target neuron would be dependent
upon whether that neuron had recently been active. This mechanism,
termed
activity-dependent
neuromodulation 8,9, allows both spatial and
temporal specificity to be obtained
from a diffuse modulatory system. The
amplification of the modulatory effects
in the paired sensory neuron would
lead to enhancement of the ability of
that sensory neuron to activate the
response system and produce the
conditioned response (Fig. 2B).
If the activity-dependent neuromodulation hypothesis for classical
conditioning is correct, then a classical
conditioning paradigm applied to individual sensory neurons in Aplysia
should specifically modify the monosynaptic connections between these
cells and the motor neurons. This was
A. H ETEROSYNAPTIC FAClLITATION
~
~
PK ~
MODULATORY
TRANSMITTER
©
cAMP
k
ATP-.~
t
Ca 2+
B. ACTIVITY-DEPENDENT NEUROMODULATION
Ca 2+
\
PK ~
cAMP,
AT[
K+
Ca 2+
Fig. 3. Model of possible molecular events contributing to heterosynaptic facilitation and activitydependent neuromodulation. (A) Heterosynaptic facilitation (which may account for sensitization).
Modulatory transmitter binding to receptor (R) activates adenylate cyclase (C) via a regulatory subunit
((7). The resulting cAMP activates one or more protein kinases (PK) whose action(s) includes closure
of steady-state K + channels. Closing K + channels results (indirectly) in increased Caz+ influx and
increased transmitter release. (B) Activity-dependent neuromodulation rwhich may be mechanism for
associative learning). An initial influx of Cae+ due to spike activity affects one or more components of
the adenylate cyclase complex. Subsequent transmitter activation of the cyclase by the modulatory
transmitter results in an ampliJ~cation of cAMP production, ultimately resulting in enhanced
transmitter release relative to that produced by the modulatory transmitter alone. Additional and
longer term effects of amplified cAMP production (mediated by increased activiation of protein
kinases) are also possible. Modified from Ref. 18.
shown in two studies that used a
classical conditioning procedure with
intracellular activation of individual
tail sensory cells and siphon sensory
cells as a neural analog of the
conditioned stimulus (CS) and shock
to the skin as the unconditioned
stimulus (US) s,9. While the sensory
neurons are normally activated by
mechanical stimulation of the skin,
artificial intracellular activation was
used to control which cells were
activated as well as the number of
action potentials and the exact timing
of the spike activity relative to the
modulatory input. The US did not
activate the sensory neurons being
examined, but did activate diffuse
neuromodulatory
input,
producing
heterosynaptic facilitation of all the
connections of the sensory neuronsS~ 9.
Paired presentation of the ~ and US
resulted in a significant enhancement
of the excitatory postsynaptic potential
481
T I N S - November 1985
compared to the facilitation produced
when the CS and US were separated in
time or when the US was presented
alone s,9. The unpaired procedure
where the CS and US were separated
in time was particularly important
because it illustrated that spike activity
and reinforcing stimuli must be temporally close in order for the associative change in synaptic efficacy to
an identical procedure applied to the
remnants of the pleural ganglia failed
to produce any pairing-specific enhancement of c A M P levels 17,18. Similar conclusions using slightly different
procedures were reached by Abrams et
al. 31.
The measurements of c A M P levels
were obtained with isolated sensory
neuron clusters and therefore the
occur.
effects that were monitored presumably occurred in the somata and not
Molecular mechanism for associative
the synaptic terminals. This effect is
information storage
interesting by itself because of the
Since enhanced cyclase activity is possibility that cyclic A M P may inbelieved to be the critical step for the fluence the synthetic capabilities of the
enhancement of synaptic efficacy that
cell soma (see below). If similar effects
underlies short-term sensitization, a were also taking place at the terminal,
further prediction of the activity- a more immediate read-out of the
dependent neuromodulation model ~ memory might be an enhancement of
for classical conditioning is that cAMP
transmitter release. A molecular
levels in a sensory neuron receiving a model is illustrated in Fig. 3. This
paired presentation of spike activity model incorporates the notion that a
and the modulatory effect of the US mechanism for the acquisition and
should be enhanced relative to a cell short-term retention of the associative
receiving unpaired stimuli.
information is simply an extension of
As a first step in testing this mechanisms already in place for nonhypothesis, sensory neurons involved associative information storage. Rein the tail-withdrawal reflex were
inforcing stimuli cause the release of
surgically isolated and exposed to a serotonin, or a related neuromodulamodification of the conditioning parator, that activates the adenylate cydigm used in the electrophysiological clase complex in the sensory neurons
studies. Rather than attempting to
(Figs 1 and 3A). At least one effect of
measure c A M P levels in a single the c A M P produced is to close resting
neuron, however, Ocorr et al. 17,18 K + channels, presumably by activating
applied a modified conditioning pro- one or more protein kinases that in
cedure to a homogeneous cluster of turn phosphorylate proteins associated
about 100 sensory neurons. A brief with K + channels 22. According to the
exposure to high K + artificial sea model for the neuronal modifications
water was used to depolarize the entire that may underly associative learning,
cluster of sensory neurons and thus
the spike activity (see below) assomimic the electrophysiological spike ciated with the CS enhances the
activity used previously as the CS. The
synthesis of cAMP in response to a
US was a brief exposure to serotonin
neuromodulator released by the US.
that was used to mimic the modulatory Therefore, even greater levels of
input normally produced by reinforc- c A M P would be produced, yielding
ing stimuli. Pairing of the stimuli was greater closure of K + channels and
accomplished by exposing isolated greater transmitter release compared
clusters of sensory neurons to the CS to the enhancement of transmitter
immediately followed by exposure to release produced by the modulator
the US. Because there are two identi- alone. Thus, increased cAMP levels
cal clusters of sensory neurons in each
seem to provide a biochemical mecanimal, contralateral clusters of sen- hanism for encoding information
sory neurons were used as controls and
about the temporal association of
received a specifically unpaired pre- separate inputs to these cells 17,18,31
sentation of the CS and US. Ocorr et and therefore may be a primary
biochemical step in the memory not
al. found that the cAMP levels of
sensory neuron clusters receiving the
only for sensitization but also for
paired presentation of the CS and US classical conditioning.
were significantly elevated relative to
A particularly intriguing question is
the levels seen in the contralateral
what aspect of spike activity is capable
control clusters receiving the unpaired
of enhancing c A M P synthesis? There
paradigm is. The effect was produced
are a number of possibilities but the
one that seems most attractive is Ca 2+
by a single pairing and appeared to be
restricted to the sensory neurons since influx. Ca 2+ influx certainly occurs
during the spike 32,33 and there are
numerous examples of interactions
between the Ca/+ and c A M P second
messenger systems 3a,35. One possible
molecular locus for the induction of
the associative change would be the
proximate and sequential interaction
of Ca 2+ and serotonin (or related
neuromodulators) with the adenylate
cyclase complex. Evidence supporting
such a Ca 2+ interaction with cAMP is
provided by studies of vertebrate brain
tissue 36 and preliminary evidence indicates the presence of a Ca2+-sensitive
cyclase in Aplysia as well 25. More
direct support of a critical role for
cyclase activity in learning has been
provided by studies of Drosophila
where it has been shown that a mutant
deficient in associative learning also
exhibits a loss of Ca2+/calmodulin
sensitivity of the particulate adenylate
cyclase. Interestingly, the mutants in
Drosophila that affect conditioning
also affect sensitization 37,38. This provides further support for the hypothesis that these two examples of learning
may share common mechanisms.
Recently, long-term changes in synaptic transmission and in the excitability of Aplysia sensory neurons
have been found in response to
simultaneous presentations of spike
activity in sensory neurons and modulatory stimuli 9A°. In view of these
persistent effects it is interesting to
speculate that the increased c A M P
content observed in sensory neuron
somata is not simply a reflection of
events occurring at the synaptic terminals. The fluctuations in the somatic
cAMP content might play a role in
triggering the long-term changes by
acting on cellular elements localized to
the soma. Such long-term changes
could involve alterations in cell metabolism or in genomic regulation and
protein synthesis brought about via
cAMP-mediated phosphorylations 34.
Altered somatic cAMP levels may not
only provide a biochemical link between short- and long-term changes in
synaptic efficacy but also between
short- and long-term memory.
The generality of activity-dependent
neuromodulation as a basic mechanism for associative learning and cellular plasticity remains to be established.
Indeed, other mechanisms are likely39'4°. It is interesting, however, that
activity-dependent neuromodulation
has been observed recently at the
crayfish neuromuscular junction al and
in hippocampal pyramidal cells - systems shown to be involved in long-
482
t e r m c h a n g e s in synaptic efficacy. It
m a y also c o n t r i b u t e to the classical
conditioning in Drosophila m e n t i o n e d
above. In addition, there is g r o w i n g
evidence f r o m a n u m b e r of v e r t e b r a t e
and i n v e r t e b r a t e animals consistent
with synergistic Ca 2+ a n d cyclic nucleotide interactions 34,35. Since Ca ~*
a n d cyclic nucleotide control s y s t e m s
are so u b i q u i t o u s , it is attractive to
think that their specific interactions
are involved in g e n e r a l m e c h a n i s m s of
synaptic plasticity and learning.
Acknowledgements
I thank V. CasteUucci, L. Cleary, T.
Crow, K. Ocorr and E. T. Waiters for
reviewing an earlier draft of this manuscript. Supported by National Institutes of
Health grant NS 19895 and Air Force
Office of Scientific Research grant AFOSR
84-213.
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Physiology and Cell Biology, University of Texas
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Medical School, P.O. Box 20708, Houston, TX
736-737
77225, USA.