Long-term sensitization training in Aplysia leads to an increase in the expression of BiP, the major protein chaperon of the ER.

Long-term memory for sensitization of the gill- and siphon-withdrawal reflexes in Aplysia californica requires RNA and protein synthesis. These long-term behavioral changes are accompanied by long-term facilitation of the synaptic connections between the gill and siphon sensory and motor neurons, which are similarly dependent on transcription and translation. In addition to showing an increase in over-all protein synthesis, long-term facilitation is associated with changes in the expression of specific early, intermediate, and late proteins, and with the growth of new synaptic connections between the sensory and motor neurons of the reflex. We previously focused on early proteins and have identified four proteins as members of the immunoglobulin family of cell adhesion molecules related to NCAM and fasciclin II. We have now cloned the cDNA corresponding to one of the late proteins, and identified it as the Aplysia homolog of BiP, an ER resident protein involved in the folding and assembly of secretory and membrane proteins. Behavioral training increases the steady-state level of BiP mRNA in the sensory neurons. The increase in the synthesis of BiP protein is first detected 3 h after the onset of facilitation, when the increase in overall protein synthesis reaches its peak and the formation of new synaptic terminals becomes apparent. These findings suggest that the chaperon function of BiP might serve to fold proteins and assemble protein complexes necessary for the structural changes characteristic of long-term memory.

The molecular biology of memory storage: a dialogue between genes and synapses.

One of the most remarkable aspects of an animal's behavior is the ability to modify that behavior by learning, an ability that reaches its highest form in human beings. For me, learning and memory have proven to be endlessly fascinating mental processes because they address one of the fundamental features of human activity: our ability to acquire new ideas from experience and to retain these ideas over time in memory. Moreover, unlike other mental processes such as thought, language, and consciousness, learning seemed from the outset to be readily accessible to cellular and molecular analysis. I, therefore, have been curious to know: What changes in the brain when we learn? And, once something is learned, how is that information retained in the brain? I have tried to address these questions through a reductionist approach that would allow me to investigate elementary forms of learning and memory at a cellular molecular level-as specific molecular activities within identified nerve cells.

A direct synaptic connection mediating both excitation and inhibition.

Neurons have generally been thought to produce only one synaptic action on any particular cell which they innervate. An identified interneuron in the abdominal ganglion of Aplysia mediates both direct excitation and inhibition to an identified follower cell. At low firing rates the interneuron produces excitatory postsynaptic potentials; however at higher firing rates these gradually diminish in size and eventually invert to inhibitory postsynaptic potentials. Electrophysiological and pharmacological evidence indicates that the connection between these cells is monosynaptic, and that a single transmitter, acetylcholine, mediates both actions. These opposite synaptic responses appear to result from the transmitter's acting on two types of postsynaptic receptors having different thresholds for activation and different susceptibilities for desensitization.

Presynaptic facilitation as a mechanism for behavioral sensitization in Aplysia.

Sensitization is an elementary form of nonassociative learning, related to behavioral arousal, in which a strong stimulus facilitates a reflex response. Studies of the neural circuit of the gill-withdrawal reflex in the isolated abdominal ganglion on Aplysia indicate that short-term sensitization is due to presynaptic facilitation. The facilitation results in a sudden increase in the amount of neurotransmitter released by the sensory neurons at their synapses with motor neurons.

Neuronal controls of a behavioral response mediated by the abdominal ganglion of Aplysia.

Tactile stimulation of the siphon and mantle shelf in Aplysia causes a characteristic withdrawal response of the external organs of the mantle cavity. A similar response also occurs spontaneously. Both responses are mediated by the abdominal ganglion and therefore provide an opportunity for correlating cellular functioning and behavior in a relatively simple and well-studied neuronal system. The withdrawal responses are controlled by five identified motor cells which receive two types of synaptic inputs. One set of excitatory connections, activated by tactile stimulation of the siphon and mantle shelf, mediates the defensive withdrawal reflex. A second set of connections is activated by a spontaneous burst of activity in a group of closely coupled interneurons which are excitatory to some of the motor cells and inhibitory to the others. This second set of connections mediates the spontaneous withdrawal response. These two inputs can therefore switch the same population of motor cells from a simple reflex to a more complex, internally organized response.

Synaptic activation of an electrogenic sodium pump.

An identified molluscan interneuron mediates different cholinergic synaptic actions by increasing the conductance of its follower cells to different ions. We have now found that this interneuron also mediates a new class of synaptic actions which does not involve a conductance change but the activation of an electrogenic sodium pump. This synaptic action results in a prolonged inhibitory synaptic potential which is dependent on metabolism and is selectively blocked by cooling and ouabain. In cells which have this synaptic potential, part of the resting membrane potential is also maintained by an electrogenic sodium pump. The same transmitter, acetylcholine, can independently stimulate both a chloride ion conductance and a sodium pump mechanism in the same follower cell by acting on two different postsynaptic receptors.

Diphasic postsynaptic potential: a chemical synapse capable of mediating conjoint excitation and inhibition.

Two identified interneurons in each buccal ganglion of Aplysia can mediate conjoined excitation and inhibition to a single follower cell. A single presynaptic action potential in one of these interneurons produces a diphasic, depolarizing-hyperpolarizing synaptic potential apparently as a result of a single transmitter acting on two types of postsynaptic receptors in the follower cell. These receptors produce synaptic potentials with differing reversal potentials, ionic conductances, time courses, rates of decrement with repetition, pharmacological properties, and functional consequences. The excitatory receptor controls a sodium conductance, the inhibitory receptor controls a chloride conductance. Both components of the synaptic potentials can be produced by iontophoretic application of acetylcholine on the cell body of the follower cell, and each component is differentially sensitive to different cholinergic blocking agents.

Neuroscience: breaking down scientific barriers to the study of brain and mind.

In this month's essay, Eric R. Kandel and Larry R. Squire chronicle how brain research has migrated from the peripheries of biology and psychology to assume a central position within those disciplines. The multidiscipline of neuroscience that emerged from this process now ranges from genes to cognition, from molecules to minds.

Requirement of a critical period of transcription for induction of a late phase of LTP.

Repeated high-frequency trains of stimuli induce long-term potentiation (LTP) in the CA1 region that persists for up to 8 hours in hippocampal slices and for days in intact animals. This long time course has made LTP an attractive model for certain forms of long-term memory in the mammalian brain. A hallmark of long-term memory in the intact animal is a requirement for transcription, and thus whether the late phase of LTP (L-LTP) requires transcription was investigated here. With the use of different inhibitors, it was found in rat hippocampal slices that the induction of L-LTP [produced either by tetanic stimulation or by application of the cyclic adenosine monophosphate (cAMP) analog Sp-cAMPS (Sp-cyclic adenosine 3',5'-monophosphorothioate)] was selectively prevented when transcription was blocked immediately after tetanization or during application of cAMP. As with behavioral memory, this requirement for transcription had a critical time window. Thus, the late phase of LTP in the CA1 region requires transcription during a critical period, perhaps because cAMP-inducible genes must be expressed during this period.

Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons.

Hippocampal long-term potentiation (LTP) is thought to serve as an elementary mechanism for the establishment of certain forms of explicit memory in the mammalian brain. As is the case with behavioral memory, LTP in the CA1 region has stages: a short-term early potentiation lasting 1 to 3 hours, which is independent of protein synthesis, precedes a later, longer lasting stage (L-LTP), which requires protein synthesis. Inhibitors of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) blocked L-LTP, and analogs of cAMP induced a potentiation that blocked naturally induced L-LTP. The action of the cAMP analog was blocked by inhibitors of protein synthesis. Thus, activation of PKA may be a component of the mechanism that generates L-LTP.

cAMP evokes long-term facilitation in Aplysia sensory neurons that requires new protein synthesis.

Behavioral sensitization leads to both short- and long-term enhancement of synaptic transmission between the sensory and motor neurons of the gill-withdrawal reflex in Aplysia. Serotonin (5-HT), a transmitter important for short-term sensitization, can evoke long-term enhancement of synaptic strength detected 1 day later. Because 5-HT mediates short-term facilitation through adenosine 3',5'-monophosphate (cAMP)-dependent protein phosphorylation, the role of cAMP in the long-term modulation of this identified synapse was examined. Like 5-HT, cAMP can also evoke long-term facilitation lasting 24 hours. Unlike the short-term change, the long-lasting change is blocked by anisomycin, a reversible inhibitor of protein synthesis, and therefore must involve the synthesis of gene products not required for the short-term change.

Long-term facilitation in Aplysia involves increase in transmitter release.

In a variety of vertebrates and invertebrates, long-lasting enhancement of synaptic transmission contributes to the storage of memory lasting one or more days. However, it has not been demonstrated directly whether this increase in synaptic transmission is caused by an enhancement of transmitter release or an increase in the sensitivity of the postsynaptic receptors. These possibilities can be distinguished by a quantal analysis in which the size of the miniature excitatory postsynaptic potential released spontaneously from the presynaptic terminal is used as a reference. By means of microcultures, in which single sensory and motor neurons of Aplysia were plated together, miniature excitatory postsynaptic potentials attributable to the spontaneous release of single transmitter quanta from individual presynaptic neurons were recorded and used to analyze long-term facilitation induced by repeated applications of 5-hydroxytryptamine. The results indicate that the facilitation is caused by an increase in the number of transmitter quanta released by the presynaptic neuron.

Sensitization in Aplysia: restoration of transmission in synapses inactivated by long-term habituation.

Long-term habituation of a simple withdrawal reflex in Aplysia leads to an inactivation of synaptic transmission between identified sensory and gill motor neurons that persists for more than 3 weeks. A single sensitizing stimulus rapidly reactivates both the depressed behavioral response and the inactivated synaptic transmission. Thus sensitization, a simple competitive form of learning, provides a mechanism whereby changing environmental demands can rapidly override the long-term memory of habituation.

Synaptic facilitation and behavioral sensitization in Aplysia: possible role of serotonin and cyclic AMP.

The neural changes accompanying sensitization of the gill-withdrawal reflex in Aplysia are associated with presynaptic facilitation at monosynaptic connections between sensory neurons and motor cells. To analyze the molecular mechanisms underlying the facilitation, the pharmacological actions of serotonin, octopamine, and dopamine were examined. Only serotonin enhanced synaptic transmission between the sensory and the motor neurons. A serotonin antagonist, cinanserin, reversibly blocked the synaptic facilitation. The action of serotonin may be mediated by adenosime 3',5'-monophosphate (cyclic AMP). Exposing the ganglion to dibutyryl cyclic AMP or injecting cyclic AMP into the cell body enhances the synaptic action of a sensory neuron. The mechanism of presynaptic facilitation, therefore, may include activation of one or more serotonergic neurons, which enhance the release of a neurotransmitter by increasing the intracellular concentration of cyclic AMP in the terminals of the sensory neurons.

Molecular biology of learning: modulation of transmitter release.

Until recently, it has been impossible to approach learning with the techniques of cell biology. During the past several years, elementary forms of learning have been analyzed in higher invertebrates. Their nervous systems allow the experimental study of behavioral, neurophysiological, morphological, biochemical, and genetic components of the functional (plastic) changes underlying learning. In this review, we focus primarily on short-term sensitization of the gill and siphon reflex in the marine mollusk, Aplysia californica. Analyses of this form of learning provide direct evidence that protein phosphorylation dependent on cyclic adenosine monophosphate can modulate synaptic action. These studies also suggest how the molecular mechanisms for this short-term form of synaptic plasticity can be extended to explain both long-term memory and classical conditioning.

Epileptogenic agents enhance transmission at an identified weak electrical synapse in Aplysia.

To examine the possibility that alterations in the effectiveness of electrical synapses might participate in epileptogenesis, the effects of several convulsants on an identified weak electrical synapse in Aplysia were examined. Application of pentylenetetrazole, strychnine, or tetraethylammonium led to a dramatic increase in the size of the electrical postsynaptic potential mediated by the synapse; penicillin was considerably less effective. In a number of animals, the increased electrical synaptic effectiveness led to the abnormal conduction of spikes across the synapse. If convulsants have a similar action in mammalian cortex, enhanced transmission at weak electrical synapses may provide abnormal pathways for the flow of seizure activity and contribute in part to the synchronous firing of neurons characteristic of epileptic activity.

Emergence of posttetanic potentiation as a distinct phase in the differentiation of an identified synapse in Aplysia.

The developmental time course of posttetanic potentiation was studied at an identified chemical synapse. In stage 11 juveniles (3 weeks after metamorphosis), the synaptic connections made by cholinergic neuron L10 onto postsynaptic neurons L2 to L6 were present but showed no posttetanic potentiation. In stage 13 adults (12 weeks after metamorphosis), the same tetanus resulted in an increase of 300 percent in the synaptic potential. A similar pattern was observed at two other identified synapses in the abdominal ganglion. Thus, the initial steps in synapse formation do not include the expression of this plastic capability. Rather, at least 10 weeks is required between the onset of synaptic function and the final expression of mature synaptic properties.

Acquisition and retention of long-term habituation in Aplysia: correlation of behavioral and cellular processes.

To examine the cellular mechanisms responsible for transition from a short-term to a long-term behavioral modification, a rapid training procedure was developed for producing long-term habituation of the defensive withdrawal of gill and siphon in Aplysia. Four ten-trial training sessions, with 1(1/2)-hour intersession intervals, produced habituation that was retained for more than 1 week. This 5-hour procedure could be applied to a test system in the isolated abdominal ganglion where the cellular changes accompanying the acquisition of long-term habituation can be examined. During acquisition, intracellular recordings were obtained from L7, a major gill and siphon motor neuron, and the pattern of stimulation used in the behavioral experiments was applied to an afferent nerve. Acquisition was associated with a progressive decrease in the complex excitatory synaptic potential produced in L7 by afferent nerve stimulation. When retention was tested 24 hours later, the synaptic decrement was still evident. Thus, a behaviorally meaningful stimulus sequence, consisting of only 40 patterned stimuli, leads to changes in synaptic effectiveness lasting one or more days in a neural pathway involved in short-term habituation of this reflex.

Target-dependent structural changes accompanying long-term synaptic facilitation in Aplysia neurons.

The mechanisms underlying structural changes that accompany learning and memory have been difficult to investigate in the intact nervous system. In order to make these changes more accessible for experimental analysis, dissociated cell culture and low-light-level video microscopy were used to examine Aplysia sensory neurons in the presence or absence of their target cells. Repeated applications of serotonin, a facilitating transmitter important in behavioral dishabituation and sensitization, produced growth of the sensory neurons that paralleled the long-term enhancement of synaptic strength. This growth required the presence of the postsynaptic motor neuron. Thus, both the structural changes and the synaptic facilitation of Aplysia sensorimotor synapses accompanying long-term behavioral sensitization can be produced in vitro by applying a single facilitating transmitter repeatedly. These structural changes depend on an interaction of the presynaptic neuron with an appropriate postsynaptic target.

Two functional effects of decreased conductance EPSP's: synaptic augmentation and increased electrotonic coupling.

Three electronically coupled motor neurons, which mediate inking behavior in Aplysia californica, receive both increased and decreased conductance excitatory postsynaptic potentials (EPSP's). The increased conductance EPSP's reduce electrical coupling among the cells, whereas the decreased conductance EPSP's increase electrical coupling. The decreased conductance EPSP's also augment the action of a previously ineffective sensory input and this augmentation is enhanced by the increase in electrical coupling. Both effects combine to trigger a stereotypic behavioral response.