Subcellular cellular and circuit mechanisms underlying classical conditioning in Hermissenda crassicornis.

THE ANATOMICAL RECORD (PART B: NEW ANAT.) 289B:25–37, 2006
REVIEW
Subcellular, Cellular, and Circuit Mechanisms
Underlying Classical Conditioning in Hermissenda
crassicornis
KIM T. BLACKWELL*
A breakthrough for studying the neuronal basis of learning emerged when invertebrates with simple nervous systems,
such as the sea slug Hermissenda crassicornis, were shown to exhibit classical conditioning. Hermissenda learns to
associate light with turbulence: prior to learning, naive animals move toward light (phototaxis) and contract their foot
in response to turbulence; after learning, conditioned animals delay phototaxis in response to light. The
photoreceptors of the eye, which receive monosynaptic inputs from statocyst hair cells, are both sensory neurons
and the first site of sensory convergence. The memory of light associated with turbulence is stored as changes in
intrinsic and synaptic currents in these photoreceptors. The subcellular mechanisms producing these changes
include activation of protein kinase C and MAP kinase, which act as coincidence detectors because they are activated
by convergent signaling pathways. Pathways of interneurons and motorneurons, where additional changes in
excitability and synaptic connections are found, contribute to delayed phototaxis. Bursting activity recorded at
several points suggest the existence of small networks that produce complex spatiotemporal firing patterns. Thus,
the change in behavior may be produced by a nonlinear transformation of spatiotemporal firing patterns caused by
plasticity of synaptic and intrinsic channels. The change in currents and the activation of PKC and MAPK produced
by associative learning are similar to those observed in hippocampal and cerebellar neurons after rabbit classical
conditioning, suggesting that these represent general mechanisms of memory storage. Thus, the knowledge gained
from further study of Hermissenda will continue to illuminate mechanisms of mammalian learning. Anat Rec (Part B:
New Anat) 289B:25–37, 2006. © 2006 Wiley-Liss, Inc.
KEY WORDS: associate learning; photoreceptors; synaptic plasticity; intrinsic excitability; coincidence detection; calcium
ASSOCIATIVE LEARNING
BEHAVIOR
Learning and Memory
Learning is “a relatively long lasting
and adaptive change in behavior re-
Dr. Blackwell received her VMD and
PhD from the University of Pennsylvania
and is an associate professor in the
School of Computational Sciences, and
the Krasnow Institute for Advanced
Studies at George Mason University.
Her research examines the synaptic and
ionic currents and second-messenger
pathways involved in associative learning, using a combination of experimental and computational techniques.
*Correspondence to: Kim T. Blackwell,
School of Computational Sciences, and
the Krasnow Institute for Advanced
Study, George Mason University, MS
2A1, Fairfax, VA 22030. Fax: 703-9934325; E-mail: avrama@gmu.edu
DOI 10.1002/ar.b.20090
Published online in Wiley InterScience
(www.interscience.wiley.com).
© 2006 Wiley-Liss, Inc.
sulting from experience” (Hall, 1976).
The breadth of species that exhibit
learning is astounding, ranging from
worms (Rankin, 2004) to humans.
Nonetheless, the capacity for learning
varies greatly among species, being
exceedingly large in primates as compared to most other mammals (Lefebvre et al., 2004). The long developmental period of primates is coupled to
learning how to survive in the environment; the expense of a long developmental period is repaid in the form
of extreme flexibility and adaptability.
Memory is intricately tied to learning,
because memory is the storage of
what we have learned. Thus, learning
is analogous to the procedure producing the change in behavior, but memory is the physical change to the organism that allows the behavior to
endure. A fascinating aspect of learning and memory is that there is a di-
chotomy in the types of learning,
which is reflected in our language. Explicit memory is the memory for facts
and events; implicit memory is the
memory for skills (Squire and Zola,
1996). When we learn facts and
events, we have it in memory, or have
memorized the information. When we
learn a skill, that too is stored in memory, but as a capability, or enhancement of skill performance.
Another intriguing aspect of learning and memory is that most of the
time, we are learning associations.
From the earliest age, an infant learns
to associate the smell of its mother
with being fed, thus the infant calms
when the mother picks it up, even
prior to being fed. Such language as
“that reminds me of . . .” and “looks
like rain,” as well as observations that
students learn information better
when it is related to what they already
26 THE ANATOMICAL RECORD (PART B: NEW ANAT.)
TABLE 1. Abbreviations
CR
CS
UR
US
AA
DAG
IP3
ERK
Conditioned Response
Conditioned Stimulus
Unconditioned Response
Unconditioned Stimulus
Arachidonic Acid
Diacylglycerol
Inositol triphosphate
Extra-cellular signal related
protein kinase
MAPK Mitogen activated protein
kinase
MEK1 ERK-activated kinase
PKA
Protein Kinase A
PKC
Protein Kinase C
PLA2
Phospholipase A2
RyR
Ryanodine Receptor
CPG Central Pattern Generator
IPSP
Inhibitory post synaptic
potential
EPSP Excitatory post synaptic
potential
know, is additional evidence of the associative nature of learning and memory.
Classical Conditioning
A breakthrough in the study of learning came with the development of an
animal model of an associative form
of learning called classical conditioning, developed by the Russian physiologist Pavlov (Windholz, 1989). Originally, Pavlov was studying glandular
secretions involved in digestion and
was inducing dogs to salivate by giving them food. The delivery of food
was preceded by a sound such as a
bell. The dogs learned that the bell
predicted the food and began salivating in response to the bell. This early
salivation response actually interfered
with the original experiments, but
Pavlov was clever enough to realize
that this was an adaptive or learned
behavior useful for studying the brain.
Continuous study during the last
century has helped to define and delineate classical conditioning (Hall,
1976). In Pavlov’s paradigm, the bell,
which initially does not evoke a response, is the conditioned stimulus
(CS); the meat, which elicits a response prior to training, is the unconditioned stimulus (US; see Table 1 for
a list of abbreviations). After repeated
presentations of the CS (sound) followed by the US (food), the sound
alone evokes a response, called the
conditioned response (CR). One of the
hallmarks of classical conditioning is
that the timing between the CS and
US presentation is critical. If the CS is
presented after the US, the animal will
never learn that the CS predicts the
US. Thus, the CS must be presented
before the US, and the time between
CS and US presentation should not be
too long (though the limiting length
depends on the species and other
characteristics of CS and US).
In-depth studies by psychologists of
factors constraining classical conditioning were designed to determine the
physical changes corresponding to
memory storage. In other words, classical conditioning was considered a
learning paradigm to determine how
the brain controls learning behavior.
Behavioral techniques coupled with
neurophysiology techniques such as extracellular recording of neuronal activity or focal brain lesions revealed that
multiple neuronal circuits participate
in this simple form of learning. One set
of sensory neuronal circuits transforms
external conditioned and unconditioned stimuli into neuronal activity
patterns. Another set of motor neuronal
circuits transforms neuronal activity
patterns into behavior. A third distinct
set of neuronal circuits stores the memory: the activity patterns of these neurons is transformed when CS and US
stimuli are presented with the correct
temporal sequence (Berger et al., 1983).
The most important question remains:
how do neurons store memories? In
other words, what are the mechanisms
whereby activity patterns of the “memory storage” neurons are transformed?
Invertebrate Classical
Conditioning
A breakthrough for studying the neuronal basis of learning emerged when
invertebrates such as Hermissenda
crassicornis were shown to exhibit
such behavior (Crow and Alkon,
1978). Hermissenda is a small nudibranch mollusk, also known as a seaslug, that lives in the coastal waters of
the Pacific Ocean, e.g., off the coast of
Monterey, California. Hermissenda
learns to associate light, the CS, with
turbulence, the US. Prior to learning,
naive animals move forward in response to light and contract their foot
in response to turbulence; after learn-
REVIEW
ing, conditioned animals contract
their foot in response to light and delay forward movement toward the
light. It is important to point out that
these are stimuli that appear in Hermissenda’s natural environment; thus,
light is a surrogate for sunlight, and
turbulence is the surrogate for wave
action. This behavior is adaptive in
that during storms, when the water is
extremely turbulent, Hermissenda
learns to avoid moving toward the
surface of the water and instead contracts its foot in order to cling to a
rock or other structure.
Many of the behavioral characteristics of Hermissenda classical conditioning are similar to those found in mammalian classical conditioning (Crow
and Alkon, 1978; Schreurs and Alkon,
2001). Hermissenda do not learn if presented with light alone, or turbulence
alone. Learning requires that the light
occurs prior to turbulence, and that turbulence follows within a few seconds.
Other behavioral properties include
contingency sensitivity (both Hermissenda and other animals learn more
slowly if unpaired stimuli are interspersed with paired stimuli) and savings (Hermissenda that have previously
learned and forgotten an association
learn more quickly the second time).
Other behavioral characteristics of classical conditioning, both those shared
with mammals and higher-order mammalian characteristics lacking in Hermissenda, are reviewed elsewhere (Matzel et al., 1998; Crow, 2004).
NEURONAL CORRELATES OF
CLASSICAL CONDITIONING
For neuroscientists, classical conditioning is a paradigm used to probe deeper
questions of how the brain stores memories. Thus, the unquestionable value in
studying classical conditioning in Hermissenda is attributed to its simple nervous system and the ability to measure
neuronal changes corresponding to the
memory trace. The central nervous system consists of paired cerebropleural
ganglia, pedal ganglia, eyes, and statocysts, the vestibular organs composed
of 13 hair cells that sense acceleration
(Fig. 1). Compared to the billions of
neurons in the mammalian brain, the
thousands of neurons in the Hermissenda brain and the stereotypical configuration of many of these neurons al-
REVIEW
THE ANATOMICAL RECORD (PART B: NEW ANAT.) 27
2003). When the light stimulus is removed, type A photoreceptors quickly
cease firing, a process known as dark
adaptation. In contrast, type B photoreceptors fire less strongly in response to
an increase in illumination, and they
also light-adapt less strongly then type
A photoreceptors (Crow, 1985; Farley et
al., 1990; Mo and Blackwell, 2003).
Thus, after the initial light response,
both type A and type B photoreceptors
respond at near equal firing frequencies. When returned to darkness, type B
photoreceptors continue to fire for prolonged periods due to their slow dark
adaptation. These different response
properties suggest different functional
roles: type A photoreceptors signal
changes in illumination, and type B
photoreceptors signal background illumination.
Photoreceptors Play Additional
Role as Memory Storage
Neurons
Figure 1. Hermissenda central nervous system. A: Sketch of seaslug showing approximate
size and central nervous system (CNS) location behind the tentacles and between the
rhinophores. B: Half of the central nervous system of Hermissenda. The photoreceptors are
visible on the periphery of the eye; in the center, they are obscured by pigment cells. The
statocyst consists of a sphere of 13 hair cells and centrally located statoconia. The optic
ganglia is a small collection of cells between the statocyst and the eye. Not pictured is the
buccal ganglia, which are connected to the pedal ganglia. Somatosensory and chemosensory inputs from tentacles and rhinophores are connected via nerves to the cerebropleural ganglia.
lows for precise investigation of
neuronal properties and neuronal networks participating in memory storage
and expression. Furthermore, it is possible to remove the entire central nervous system and apply the same paired
stimuli of classical conditioning to this
in vitro nervous system. Thus, the in
vitro preparation allows scientists to
probe the inner workings of neurons
while memories are being stored.
Photoreceptors Comprise
Sensory Neurons for CS
Each of the Hermissenda eyes has three
type B and two type A photoreceptors
(Fig. 2A) (Stensaas et al., 1969). The
photoreceptors are sensory cells for the
CS stimulus and transduce light energy
into depolarization. The type A and type
B photoreceptors exhibit characteristic
differences reminiscent of the difference between mammalian rods and
cones: type B photoreceptors are sensitive to dimmer lights than are type A
photoreceptors (Alkon and Fuortes,
1972; Mo and Blackwell, 2003). The
temporal response to light stimuli also
differs (Fig. 3A). Type A photoreceptors
respond to an increase in illumination
with a rapid increase in firing frequency, followed by rapid light adaptation (Crow, 1985; Farley et al., 1990;
Yamoah et al., 1998; Mo and Blackwell,
Decades of work by Alkon and colleagues (Crow and Alkon, 1978) have
revealed that the photoreceptors are
the first site of convergence of the CS
and US stimuli and a key locus of
memory storage. The sensory cells for
the US are the statocyst hair cells,
which transduce turbulence or gravitational energy into depolarization
(Detwiler and Fuortes, 1975). Statocyst hair cells release the inhibitory
neurotransmitter GABA (Alkon et al.,
1993) onto the terminal branches of
the photoreceptors. Thus, during classical conditioning, paired CS and US
stimuli produce depolarization and
GABA receptor activation of the photoreceptors. When light and turbulence are presented in the correct
temporal sequence, intracellular processes are activated and produce intrinsic changes to the photoreceptors.
Thus, the memory of the association
is stored as a change in response properties of these photoreceptors. Interestingly, type A and B photoreceptors
are modulated differently by classical
conditioning paradigms.
Intracellular recordings from animals that have been classically conditioned, or from applying a classical
conditioning procedure to the in vitro
nervous system, have revealed several
changes in photoreceptor properties
28 THE ANATOMICAL RECORD (PART B: NEW ANAT.)
Figure 2. Network of Hermissenda photoreceptors. A: Each eye has two type A and three
type B photoreceptors. The type B photoreceptors mutually inhibit each other. The type B
photoreceptors also inhibit the type A photoreceptors. Several ionic channels are shown
schematically. B: Classical conditioning causes a decrease in conductance of several ionic
channel in the type B photoreceptors (narrower channels) and an increase in conductance of the delayed rectifier potassium channel in the type A photoreceptors (wider
channels). In addition, the inhibition from type B to type A photoreceptors is increased; the
effect of mutual inhibition among type B photoreceptors is increased due to the increase
in input resistance.
that are correlated with learning behavior, or the presentation of paired
stimuli, respectively. Type B photoreceptors show an increase in excitability due to the conditioning procedure,
as compared with type B photoreceptors, which received the unpaired
training procedure (Fig. 3C and D).
The increase in excitability is exhibited as an increase in input resistance,
an increase in the firing frequency due
to light stimulation or current injection (Crow and Alkon, 1980; Matzel
and Rogers, 1993; Blackwell and
Alkon, 1999), and an enhanced longlasting depolarization following light
stimulation (Alkon and Grossman,
1978). The increased excitability is accompanied by a reduction of the transient potassium current, the calciumdependent potassium current, and the
persistent calcium current (Alkon et
al., 1985; Collin et al., 1988). In contrast, type A photoreceptors exhibit a
decrease in excitability (Farley et al.,
1990; Frysztak and Crow, 1993). This
includes a decrease in the generator
potential, and a decrease in input resistance, which is accompanied by an
increase in the delayed rectifier potassium current. The effect of classical
conditioning on firing frequency of
type A photoreceptors is less clear,
with reports of increase (Frysztak and
Crow, 1993) and decrease (Farley et
al., 1990; Farley and Han, 1997).
REVIEW
The most exciting aspect of these
discoveries is that they may represent
general mechanisms of memory storage (Matzel et al., 1998; Schreurs and
Alkon, 2001; Daoudal and Debanne,
2003). Long-term changes in excitability have been demonstrated in
rabbits consequent to classical conditioning (Moyer et al., 1996; Schreurs
et al., 1998). An accompanying reduction in afterhyperpolarization suggests that the increase in excitability is
mediated by a reduction in potassium
currents (Coulter et al., 1989; Schreurs et al., 1998). Additional evidence
for the role of potassium currents
comes from genetic studies of Drosophila. Alteration of shaker transient
potassium channels or eag potassium
channels produces deficits in conditioning (Cowan and Siegel, 1986).
Thus, though memory storage in sensory neurons may be unusual, the
changes in potassium currents and excitability in neurons receiving conver-
Figure 3. Electrical activity of type A and B photoreceptors. A: Simulated response to
30-msec duration light. Type A photoreceptors give a brief burst of spikes and then quickly
dark-adapts. Type B photoreceptors respond with initially lower firing frequency than type
A photoreceptors, but continue to fire for a long time in the dark. B: Response of type B
photoreceptors to in vitro classical conditioning. During the first pairing of 3-sec light and
2-sec turbulence, the response looks similar to light alone, with a sustained firing for many
seconds after light offset. After the fourth pairing, burst firing appears. C and D: Response to
current injection before (unpaired) and after classical conditioning. Type B photoreceptors
develop an increase in spike activity and an increase in input resistance. This change in
activity requires a decrease in the transient potassium current, calcium-dependent potassium current, calcium current, and leak potassium current. Type A photoreceptors decrease their spike activity. This change is mediated by an increase in the delayed rectifier
potassium current.
REVIEW
gent inputs is likely to be a general
mechanism of memory storage.
Photoreceptor Synaptic
Interactions Contribute to
Memory Trace
Another mechanism underlying classical conditioning in Hermissenda is
changes in synaptic interactions
among photoreceptors. As illustrated
in Figure 2A, the type B photoreceptors are mutually inhibitory, and the
type A photoreceptors are inhibited by
the type B photoreceptors (Fig. 2A)
(Alkon and Fuortes, 1972; Goh and
Alkon, 1984). Classical conditioning
enhances the inhibition of medial type
A photoreceptors by medial type B
photoreceptors (the IPSP is enhanced), but there is no change in inhibition of lateral type A by lateral
type B photoreceptors (Frysztak and
Crow, 1997). In contrast, in vitro conditioning produces an enhancement
of IPSPs from type B to type A photoreceptors, both medial and lateral
(Fig. 2B) (Schuman and Clark, 1994).
There have been no reports of changes
in synaptic strength of inhibitory connections between pairs of type B photoreceptors, but the increased input
resistance and firing frequency would
produce enhanced inhibition regardless of whether the synapse itself was
modulated. Furthermore, action potential width in type B photoreceptors
is increased (Gandhi and Matzel,
2000), which may be accompanied by
an increase in calcium influx and consequent increase in release probability. In summary, classical conditioning potentiates synaptic interactions
not only by synaptic mechanisms, but
also secondary to the modulation
of voltage-dependent channels and
membrane properties.
Long-term synaptic potentiation,
specifically that produced by synaptic
mechanisms, has long been studied as
a mechanism of memory storage in
mammals (Bliss and Collingridge,
1993; Malinow and Malenka, 2002).
Despite decades of study, the connection between memory and hippocampal LTP is tenuous (Teyler and DiScenna, 1984; Shors and Matzel, 1997).
Perhaps the strongest evidence for
synaptic plasticity as a mechanism of
memory storage is observed in the ba-
THE ANATOMICAL RECORD (PART B: NEW ANAT.) 29
solateral nucleus of the amygdala. In
vivo recordings in rats reveal synaptic
potentiation after fear conditioning
but not in unpaired controls (Blair et
al., 2001). In mammals, addressing
how synaptic plasticity produces a
change in behavior is inaccessible due
to the large number of neurons and
synapses involved in producing behavior. In contrast, further studies of
Hermissenda may reveal scalable
mechanisms whereby changes in synaptic plasticity produce changes in activity patterns that control behavior.
Modulation of Ionic and
Synaptic Channels Produce
Change in Spatiotemporal
Firing Patterns
How is modulation of ionic and synaptic channels translated into a change in
behavior? A novel approach to this
question involves considering the five
photoreceptors as a small neuronal network (Fost and Clark, 1996b). The light
response of this network constitutes a
spatiotemporal firing pattern that is
shaped by inhibitory interactions among
photoreceptors. Modulation of both
synaptic and ionic currents transforms
the spatiotemporal pattern in a nonlinear manner due to the inhibitory synaptic interactions. In this manner, the
increase in variance of the interspike
interval due to classical conditioning
(Crow, 1985) may be due to the nonlinear transformation from tonically firing
to periodic burst firing (Fig. 3B) (Fig. 8
of Mo and Blackwell, 2003).
Periodic burst firing patterns are seen
in many systems, such as networks with
mutually inhibitory interactions and sustained excitatory inputs (Popescu and
Frost, 2002), and networks of coupled excitation and inhibition (Warren et al.,
1994). Both of these network configurations are present in the visual system of
Hermissenda. Mutual inhibition with
excitation is present in the three mutually
inhibitory type B photoreceptors; tonic
excitatory input is provided by the
light-induced long-lasting depolarization
(Alkon and Grossman, 1978). Coupled
excitation and inhibition is seen in connections between type B cells and optic
ganglion cells: the photoreceptors inhibit
optic ganglion cells, and optic ganglion
cells excite type B photoreceptors (Alkon,
1980a). Both strength and persistence of
neuronal synaptic interactions influence
the occurrence and characteristics of periodic activity (White et al., 2000). Thus,
periodic bursting may develop after classical conditioning due to the increased
strength of inhibitory interactions among
type B photoreceptors.
Computational Models
Investigate Relationship
Between Ionic Channels and
Neuronal Activity
The Hermissenda community has a
strong tradition of computational
modeling to integrate and synthesize
results from myriad experiments into
a coherent view of neuronal memory
storage. The ultimate goal with such
models is to demonstrate how paired
stimuli produce changes in neuronal
properties, neuronal activity, and motor output. An additional advantage of
computational models is to explicate
and evaluate the assumptions that always accompany conceptual models.
Presently, computational models of
Hermissenda photoreceptors have
been used primarily to investigate two
issues. The first is the second-messenger mechanisms underlying temporal
sensitivity (discussed in the next section), and the second is the mechanisms (ionic currents) underlying the
various changes in excitability and
synaptic efficacy.
Several models were created to test
hypotheses regarding the role of ionic
currents in producing the enhanced
generator potential, increased spike
frequency, synaptic potentiation, and
increase in input resistance. These
models include voltage-dependent,
calcium-dependent, and light-induced
currents. The first model (Sakakibara
et al., 1993) supported the hypothesis
that a reduction in the transient and
calcium-dependent potassium current
produces an increased generator potential and enhanced long-lasting depolarization. Later models confirmed
these results and in addition showed
that the reduction in the calcium-dependent potassium current produced
a greater influence on the plateau potential and light-induced firing frequency than did the transient potassium current (Fost and Clark, 1996a).
A reduction in the persistent calcium
current and an enhancement in the
30 THE ANATOMICAL RECORD (PART B: NEW ANAT.)
hyperpolarization-activated current,
two other ionic current changes correlated with classical conditioning,
tended to oppose each other in terms
of effect on plateau potential and firing frequency (Cai et al., 2003).
The enhancement of synaptic connections is more difficult to explain
and may be produced by mechanisms
distinct from the changes in voltagedependent currents. The models
above demonstrate that the reduction
in the transient potassium current
produces spike broadening (Fost and
Clark, 1996a; Flynn et al., 2003). Since
neurotransmitter release depends on
calcium influx, which depends on
spike width, this result suggests that a
reduction in the transient potassium
current produces synaptic facilitation.
Nonetheless, when the reduction in
transient potassium current is coupled with a reduction in calcium current, not only is the enhanced calcium
influx eliminated, but calcium influx
is depressed below the control (preconditioned) value. Furthermore, an
enhancement in the hyperpolarization activated current does not compensate for the reduced calcium influx, most likely due to inactivation of
this current at depolarized potentials.
Thus, these four current changes result in a reduction in calcium influx,
suggesting that the synaptic facilitation is produced by a mechanism independent of spike broadening, such
as modulation of postsynaptic channels or presynaptic release probability.
Computational Models Suggest
a Role for Light-Induced
Potassium Current
In all of these models, the enhanced
long-lasting depolarization not only
requires a reduction in the transient
and calcium-dependent potassium
currents, but also involves the lightinduced potassium current. Specifically, the baseline long-lasting depolarization, observed in untrained
Hermissenda after light offset (Alkon
and Grossman, 1978), is produced by
the light-induced potassium current
(Detwiler, 1976; Alkon et al., 1984;
Blackwell, 2002b, 2004), which has
properties similar to leak potassium
currents (Buckler, 1999; Donnelly,
1999). This baseline depolarization
activates both the transient and calcium-dependent potassium currents,
which are inactivated at resting potential due to their voltage dependence. A
reduction in the transient and calcium-dependent potassium currents
does not produce an enhanced depolarization in a model lacking the lightinduced potassium current (Blackwell, 2004).
The light-induced potassium current also may be required to explain
the increase in input resistance measured with hyperpolarizing current
injection. A decrease in the transient
and calcium-dependent potassium
currents is not sufficient because
these currents are not active at resting
potential. Similarly, an enhancement
in the hyperpolarization-activated
current, which is active at resting potential, actually decreases the input
resistance at hyperpolarized potentials. One possibility, which needs to
be evaluated experimentally, is that
the light-induced potassium current is
modified by classical conditioning.
Since this current is normally open
and conducting at rest, a reduction in
this current (either a decrease in total
conductance or a decrease in open
probability or duration) would decrease total membrane conductance
and increase input resistance at all
membrane potentials (Fig. 3C and D).
SUBCELLULAR MECHANISMS FOR
TEMPORAL SENSITIVITY
This research in Hermissenda, together with overwhelming evidence
from other model systems (Matzel et
al., 1998; Kandel and Pittenger, 1999),
demonstrates that long-term memory
storage consists of changes in ionic
and synaptic currents in individual
neurons. This naturally leads to a
more intriguing question: What are
the subcellular events that lead to
modulation of ionic currents when
paired stimuli are presented in correct
temporal sequence? In other words,
what are the subcellular mechanisms
that are sensitive to temporal pattern
in the photoreceptors of Hermissenda? The requirement for paired
stimuli in the behavioral paradigm
naturally translates into a requirement for either two different intracel-
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lular messengers or two different
sources of a single intracellular messenger molecule.
Elevation in Calcium Required
for Memory Storage
In Hermissenda photoreceptors, one
of the first intracellular messengers
identified as meeting this requirement
was calcium. Both light stimulation
and depolarization produced elevations in calcium via voltage-dependent calcium channels (Connor and
Alkon, 1984) and release from intracellular stores (Berridge and Galione,
1988; Muzzio et al., 1998). The rebound depolarization observed after
relief of hair cell inhibition, along
with the progressively enhanced depolarization and calcium elevation
noted during in vitro training, led to
the hypothesis that temporal sensitivity was attributed to the requirement
for two sources of calcium: one in response to light, and the second due to
depolarization (Alkon, 1980b). This
hypothesis was supported by the observation that a calcium elevation was
required for the enhanced excitability
of type B photoreceptors. Blocking
the elevation in calcium (Matzel and
Rogers, 1993), or even just one of the
calcium sources (Talk and Matzel,
1996; Blackwell and Alkon, 1999), prevented these correlates of memory
storage.
Activation of PKC Is Involved
in Memory Storage
Protein kinase C (PKC) was the second intracellular molecule identified
as playing a critical role in classical
conditioning. Classical conditioning
produces a translocation of PKC from
the cytosol to the membrane (Muzzio
et al., 1997) and PKC phosphorylates
the transient and calcium-dependent
potassium channels, decreasing their
maximum conductance and producing an increased input resistance and
evoked spike frequency (Neary et al.,
1981; Farley and Auerbach, 1986).
More importantly, PKC requires two
or more intracellular messengers for
activation: transient activation requires an elevation in calcium and diacylglycerol (Asaoka et al., 1988; Oancea and Meyer, 1998), both of which
are produced by light stimulation
(Talk and Matzel, 1996; Talk et al.,
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THE ANATOMICAL RECORD (PART B: NEW ANAT.) 31
1997; Sakakibara et al., 1998). Persistent activation of PKC requires not
only calcium and diacylglycerol, but
also arachidonic acid (Lester et al.,
1991; Shinomura et al., 1991), which
is produced by phospholipase A2. Prevention of in vitro classical conditioning with an inhibitor of phospholipase
A2 (Talk et al., 1997) supports the role
of arachidonic acid in classical conditioning.
Since light alone produces transient
activation of PKC (but does not produce classical conditioning), then turbulence-induced hair cell activity
must somehow contribute to activation of PLA2 and persistent activation
of PKC (Fig. 4). Either GABAB receptors are directly coupled to PLA2, or
GABAB stimulation produces an elevation in calcium, which combines
with the light-induced calcium, to
produce a larger or more prolonged
calcium elevation that activates PLA2
(Hirabayashi et al., 1999). The possibility that GABAB stimulation contributes to the calcium elevation in the
soma (independent of the effect on
membrane potential) was suggested
by the observation of a calcium wave
propagating from the terminal
branches to the soma (Ito et al., 1994).
Moreover, dantrolene, which prevents
the propagation of calcium waves,
prevents in vitro classical conditioning of Hermissenda (Blackwell and
Alkon, 1999).
PLA2 Mediates Contribution of
GABA Stimulation to PKC
Activation
These contributions to the calcium elevation were tested both experimentally and using model simulations.
Model simulations demonstrated that
the rebound depolarization produced
an enhanced calcium elevation and a
cumulative depolarization only when
light and hair cell stimulation occur in
the correct temporal relationship
(Werness et al., 1993). However, subsequent experiments showed that a
cumulative depolarization was not
necessary to produce the enhanced
calcium (Matzel and Rogers, 1993).
The alternative, that GABA stimulation contributes to the calcium elevation in the soma independent of the
effect on membrane potential, was
Figure 4. Subcellular mechanisms underlying classical conditioning. Molecules are colorcoded as follows: receptors are magenta; membrane permeable messengers are pink;
membrane bound enzymes are yellow; diffusible messengers are light green; calcium
release channels are dark green; enzymes ultimately responsible for long-term memory
storage are light blue. Solid lines indicate known pathways; dashed lines indicate hypothesized or multistep pathways. Light activates rhodopsin and a second-messenger cascade
that produces an increase in DAG and calcium. GABAB receptor stimulation produces an
increase in arachidonic acid either by direct activation of PLA2 or by calcium activation of
PLA2 (with PLC and IP3 as intermediates). Serotonin leads to an increase in MEK and ERK
phosphorylation, though the G-proteins and other messengers involved have not been
identified. Possible mechanisms of temporal sensitivity include persistent PKC activation
requiring light-induced DAG and turbulence (GABA)-induced AA, or ERK activation requiring light-induced transient PKC activation and turbulence (serotonin)-induced MEK
activation.
evaluated subsequently. Model simulations demonstrated that, if metabotropic GABAB receptors are coupled
to phospholipase C, then the GABA
stimulation alone initiates a calcium
elevation that propagates as a wave
(via release through ryanodine receptors) toward the soma (Blackwell,
2002a). However, inactivation of the
ryanodine receptor by the light-in-
32 THE ANATOMICAL RECORD (PART B: NEW ANAT.)
duced calcium wave propagating
from the rhabdomere toward the terminal branches produces a refractory
period and prevents the GABA-induced calcium wave from propagating past the light-induced calcium
wave when the stimuli are paired
(Blackwell, 2004). Thus, the CS and
US waves destructively interfered
with each other, preventing the USinduced calcium from adding to the
CS-induced calcium. This result suggested that a different mechanism activated by hair cell activity combines
with the light-induced calcium elevation to initiate memory storage.
A more likely possibility is that
GABAB receptors are directly coupled
to PLA2 and that GABA released onto
the terminal branches leads to production of arachidonic acid. Application of GABA produces an increase in
arachidonic acid, comparable to that
produced by direct stimulation of
PLA2 (Muzzio et al., 2001). Application of an arachidonic acid analogue
plus light produces an increase in excitability similar to that seen with
classical conditioning, and it is
blocked by the PKC inhibitor chelerythrine. Presently, this hypothesis
has the most support, but confirmation requires delineation of the G-proteins that couple GABAB receptors to
PLA2.
Serotonin-Activated Pathways
Contribute to Memory Storage
Yet another possibility is that hair
cells activate interneurons that release
serotonin onto the photoreceptor
soma (Land and Crow, 1985). Serotonin is synthesized and released in the
Hermissenda nervous system (Auerbach et al., 1989), and type B photoreceptors depolarize when serotonin
is applied to the soma (Rogers and
Matzel, 1995). Light paired with serotonin has been shown to be an effective in vivo training paradigm, producing a reduction in phototaxis
similar to that observed with standard
classical conditioning paradigms
(Crow and Forrester, 1986). Light
paired with serotonin leads to an enhanced light response (Crow and
Bridge, 1985) and a PKC-dependent
increase in both input resistance and
excitability of the type B photoreceptor (Crow et al., 1991). The modula-
tion of ionic channels accompanying
these changes also are similar to that
observed with paired light and hair
cell stimulation (Crow and Bridge,
1985; Farley and Wu, 1989; AcostaUrquidi and Crow, 1993). Despite all
of this evidence, no one has demonstrated serotonergic projections onto
the photoreceptors. Thus, support for
serotonin as a critical neurotransmitter requires identifying the neurons
that release serotonin due to hair cell
stimulation.
Whereas GABAB stimulation produces arachidonic acid that is required for persistent activation of
PKC, the precise role of serotonin in
the activation of second-messenger
systems is poorly understood. Conditioning using light paired with rotation or serotonin induces the activation of ERK1 and ERK2, members of
the mitogen-activated protein kinase
(MAPK) family (Crow et al., 1998), by
ERK activating kinase (MEK1; Fig. 4),
via both PKC-dependent and PKC-independent pathways (Crow et al.,
2001). In mammals (Sweatt, 2001),
MAPK kinase is activated by an
MAPK kinase kinase such as Raf-1 or
B-Raf, which are activated (directly or
via intermediaries) by PKC, protein
kinase A (PKA), or growth factor tyrosine kinase receptors. In Aplysia, serotonin receptors are coupled to PKA
activation and subsequent ERK activation (Barbas et al., 2003). Whether
PKA or tyrosine kinase is involved in
ERK activation in Hermissenda photoreceptors and the role of ERK in
temporal sensitivity await further
study.
CIRCUITRY
Classical conditioning leads to a
change in the spatiotemporal firing
pattern of the set of five photoreceptors, which is further transformed
into a qualitative change in motorneuron activation by interactions within
interneuron layers. Which neurons
are involved, and how do they interact
to transform the spatiotemporal firing
pattern into a change in behavior?
Many of the relevant interneurons in
the cerebropleural ganglia and motorneurons in the pedal ganglia have
been characterized.
REVIEW
Light-Sensitive Interneurons
Receive Mono- and
Polysynaptic Input From
Photoreceptors
One layer of interneurons in the cerebropleural ganglia, which are spontaneously active, are called central visual neurons or type I interneurons
(Crow and Tian, 2002b). These interneurons receive either EPSPs (type ␣
or Ie) or IPSPs (type ␤ or Ii) through a
direct synaptic connection from ipsilateral type B photoreceptors. These
interneurons also receive hair cell inputs of the same sign as type B photoreceptor inputs, i.e., interneurons
that received EPSPs from type B photoreceptors also received EPSPs from
hair cells (Fig. 5). Note that the connections from photoreceptors to type
I interneurons is not indiscriminate
and convergent, but highly targeted
and divergent. Each photoreceptor
makes connections to several type I
interneurons, but each type I interneuron receives inputs from only a
single photoreceptor. Thus, lateral
and medial photoreceptors do not
project to the same interneurons, nor
do type A and type B photoreceptors
project to the same interneurons.
A second layer of interneurons are
light-sensitive but do not receive
monosynaptic projections from photoreceptors. These spontaneously active neurons are called type II interneurons. The type IIe interneurons
receive polysynaptic excitatory input
from photoreceptors and project primarily to the ipsilateral pedal ganglion. The type IIi interneurons receive polysynaptic inhibitory input
from photoreceptors and project to
unidentified neurons in the contralateral cerebropleural ganglion (Crow
and Tian, 2002b).
Role of Dorsal Motorneurons in
Delayed Phototaxis
The first set of light-responsive motorneurons studied are located on the
dorsal surface of the pedal ganglia.
These motorneurons are spontaneously active in the dark. Some of the
motorneurons (MN1, P9, some MN4)
increase their activity to light; others
(P7, some MN4) decrease their activity in response to light (Richards and
Farley, 1987; Hodgson and Crow,
REVIEW
THE ANATOMICAL RECORD (PART B: NEW ANAT.) 33
Figure 5. Interneurons and motorneurons involved in phototaxis and foot contraction. Only
established monosynaptic connections are illustrated. The postsynaptic targets of the IIi
and IIe interneurons have not been identified, thus the connections only indicate whether
the projections are ipsilateral or contralateral. Only a single type A photoreceptor, type B
photoreceptor, hair cell, Ii interneuron, and Ie interneuron are illustrated. In reality, the
multiple photoreceptors project to unique layer I interneurons.
1991). Of these, MN1 is thought to be
the most significant, because stimulation causes movement of the tail, and
because it receives polysynaptic excitatory input (via a type 1 interneuron)
from type A photoreceptors (Goh and
Alkon, 1984).
The first hypothesis regarding the
expression of classical conditioning
was based on the differences in these
motorneurons between paired and
randomly trained Hermissenda. The
light-induced increase in activity of
MN1 and P9 is smaller in paired animals (Goh et al., 1985; Hodgson and
Crow, 1992). Similarly, the light-induced decrease in activity of P7 is
smaller in paired animals. Prior to
conditioning, type A photoreceptors
indirectly excite motor neurons, such
as MN1, responsible for positive phototaxis (Fig. 6, left: gray connections).
After conditioning, an increase in excitability of type B photoreceptors
leads to an increase in type B to type A
photoreceptor inhibition, which leads
to a decrease in excitation from type A
photoreceptors to MN1 (and P9) motorneurons (Fig. 6, right: gray connec-
tions), which results in less movement
toward the light and delayed phototaxis.
This hypothesis is consistent with
the effect of classical conditioning on
pedal nerve activity and delayed phototaxis. Locomotion is controlled by
four of the six pedal nerves (Richards
and Farley, 1987) through which all
motorneuron axons travel. The multiunit pedal nerve activity of control animals increases by about 10 –20% during light stimulation; in paired
animals, the multi-unit activity increases less (Rogers and Matzel, 1996)
or decreases below the dark activity
level (Richards and Farley, 1987).
These observations are consistent
with the hypothesis that type B photoreceptors suppress pedal nerve activity via suppression of type A photoreceptors, but do not explain foot
contraction that precedes delayed
phototaxis.
Figure 6. Information flow from sensory neurons to motorneurons involved in classical
conditioning behavior. Polysynaptic connections are indicated by dashed lines. Some of
the layer I interneurons have been removed for clarity. T1 indicates the type 1 interneuron
described by Goh and Alkon (1984). The change in line thickness on the post-conditioning
side of the figure indicates whether the connection has an increase in strength (due to
either an increase activity of the presynaptic neuron or an increase in synaptic weight) or
a decrease in strength. Pathways are color-coded by their effect on motor neurons. Gray,
excitation by type A; green, excitation by type B; red, disinhibition by type B, becomes
stronger with conditioning; blue, inhibition by type B, becomes stronger with conditioning;
black, UR pathway. The excitation of MN1 and P9 is smaller after classical conditioning. In
addition, the increase in inhibition of VP1 and VP2 via the Ie interneuron is stronger than the
increase in disinhibition via the IIi and IIe interneurons, resulting in a net decrease in
light-induced activity of VP1 and VP2 after classical conditioning.
34 THE ANATOMICAL RECORD (PART B: NEW ANAT.)
Ventral Motorneurons Are
Involved in Classical
Conditioning Behavior
Recently, Crow and colleagues have
discovered additional circuits of information flow involved in classical conditioning behavior. They have characterized a second set of motorneurons
on the ventral surface of the pedal
ganglia, as well as additional interneurons receiving input from statocyst hair cells. VP1 and VP2 are motor
neurons located on the ventral surface
of the pedal ganglia and are responsible for ciliary movement mediating
phototaxis (Crow and Tian, 2003a).
VCMN is a foot contraction motorneuron located on the ventral surface
of the pedal ganglia. Several interneurons directly and indirectly project to
these ventral motorneurons. Firing of
both Ii and IIi interneurons is accompanied by an increase in IPSPs in VP1
and VP2; thus, these neurons have an
indirect inhibitory projection to the
ciliary motorneurons. In contrast, firing of the interneuron IIe produces a
decrease in IPSPs to VP1 and VP2,
implying that IIe is inhibiting a spontaneously active neuron that provides
tonic inhibition to VP1 and VP2. Such
a neuron may be the IIIi interneuron,
which inhibits VP1 and VP2 (Fig. 5).
All three of these pathways (Fig. 6, red
connections) produce polysynaptic
disinhibition of VP1 and VP2 motorneurons in response to light. Alternatively, the pathway through interneuron Ie is polysynaptic inhibition (Fig.
6, blue connections), because firing of
interneuron Ie produces an increase
in IPSPs to VP1 and VP2. The US
pathway is mediated by VCMN, which
does not receive input from interneurons that receive polysynaptic inputs
from photoreceptors (Crow and Tian,
2004). It receives excitatory input
from the spontaneously active Ib interneuron (Fig. 5), which is indirectly
excited by statocyst hair cells (Fig. 6,
black connections). Thus, via this
pathway, vestibular signals are converted into foot contraction.
Changes in excitability and synaptic
connection strength in this network
can further explain the change in phototaxis caused by classical conditioning. First, paired training causes facilitation of the monosynaptic PSP
measured in type I interneurons and
an increase in light-evoked spike activity (Crow and Tian, 2002a). The enhancement of complex EPSPs to interneuron Ie and complex IPSPs to
interneuron Ii is partly due to the increase in type B photoreceptor spike
frequency and partly due to the enhancement in intrinsic properties of
the interneuron. This enhancement in
the Ie light response is propagated
through the circuit as an increase in
inhibition of VP1 (Crow and Tian,
2003b). Presumably, this enhancement of inhibition is greater than the
enhanced disinhibition (caused by the
increased type B activity) mediated by
the IIe and IIi interneurons. The net
result is that prior to pairing, light
causes VP1 excitation via disinhibition, whereas after pairing light
causes VP1 inhibition. In summary,
the change in VP1 response consequent to classical conditioning can explain both the change in pedal nerve
multi-unit activity and the change in
phototaxis; however, the neural circuitry and information flow that
explains the foot contraction in response to light after classical conditioning remains concealed.
Do Central Pattern Generators
Play a Role in Hermissenda?
In other systems, many motor behaviors are controlled by neuronal activity patterns produced by an interconnected sets of interneurons called the
central pattern generator (CPG). The
CPG activates motorneurons to produce motor behavior; an alteration in
the neuronal activity pattern of the
CPG transforms the motor behavior
(e.g., Combes et al., 1999; Nargeot et
al., 1999). The pattern may be altered
by a change in synaptic inputs, e.g.,
due to sensory inputs, or neuromodulators, which modify synaptic or ionic
channels in CPG neurons (Marder and
Calabrese, 1996). For example, in mollusks such as Tritonia and Pleurobranchia, both crawling and escape
swimming are produced by central pattern generators (Jing and Gillette, 1999;
Popescu and Frost, 2002). The rhythmic motor pattern is switched from
crawling to escape swimming by patterns of synaptic inputs from command
neurons (Jing and Gillette, 2000).
The periodic burst firing of pedal
nerves suggests that Hermissenda lo-
REVIEW
comotion is controlled by central pattern generators homologous to those
in Tritonia and Pleurobranchia. The
change in photoreceptor spatiotemporal firing pattern caused by classical
conditioning may produce a nonlinear change in the rhythmic motor pattern from that producing phototaxis
to that producing foot contraction.
Experiments to fill in the missing connections in Figure 5 may discover
such a central pattern generator and
demonstrate for the first time the
complete circuitry and activity patterns responsible for classical conditioning behavior.
CONCLUSION
The study of Hermissenda classical
conditioning was initiated decades
ago in the belief that uncovering the
secret to learning and memory had
the best chance of success by studying
simple learning behaviors in simple
animals. Indeed, a body of research
convincingly demonstrates that ionic
and synaptic channels in photoreceptors are modified during memory storage. Similarly, many of the secondmessenger pathways activated by CS
and US have been delineated. Nonetheless, this review argues that significant gaps in knowledge exist, most
notably (but not exclusively) regarding the neural circuitry translating
changes in photoreceptor excitability
to changes in behavior. The complexity of memory storage mechanisms revealed thus far, as well as the parallels
with mammalian systems, reinforces
the imperative to investigate invertebrate learning and memory.
ACKNOWLEDGMENTS
The author thanks Harold Morowitz
for comments on an earlier version of
the manuscript. The author apologizes to the many authors whose work
was not cited; the references had to be
limited due to editorial discretion.
Parts of the research reported in the
manuscript was supported by the National Science Foundation (NSF) and
the National Institute of Mental
Health (NIMH).
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