Overexpression of and RNA interference with the CCAAT enhancer-binding protein on long-term facilitation of Aplysia sensory to motor synapses

In the marine mollusk Aplysia, the CCAAT/enhancer-binding protein, ApC/EBP, serves as an immediate early gene in the consolidation of long-term facilitation in the synaptic connection between the sensory and motor neurons of the gill-withdrawal reflex. To further examine the role of ApC/EBP as a molecular switch of a stable form of long-term memory, we cloned the full-length coding regions of two alternatively spliced forms, the short and long form of ApC/EBP. Overexpression of each isoform by DNA microinjection resulted in a l6-fold increase in the expression of the coinjected luciferase reporter gene driven by an ERE promoter. In addition, when we overexpressed ApC/EBP in Aplysia sensory neurons, we found that the application of a single pulse of 5-HT that normally induced only short-term facilitation now induced long-term facilitation. Conversely, when we attempted to block the synthesis of native ApC/EBP by microinjecting double-strand RNA or antisense RNA, we blocked long-term facilitation in a sequence-specific manner. These data support the idea that ApC/EBP is both necessary and sufficient to consolidate short-term memory into long-term memory. Furthermore, our results suggest that this double-strand RNA interference provides a powerful tool in the study of the genes functioning in learning and memory in Aplysia by specifically inhibiting both the constitutive and induced expression of the genes.     

Multiple serotonergic mechanisms contributing to sensitization in aplysia: evidence of diverse serotonin receptor subtypes

The neurotransmitter serotonin (5-HT) plays an important role in memory encoding in Aplysia. Early evidence showed that during sensitization, 5-HT activates a cyclic AMP-protein kinase A (cAMP-PKA)-dependent pathway within specific sensory neurons (SNs), which increases their excitability and facilitates synaptic transmission onto their follower motor neurons (MNs). However, recent data suggest that serotonergic modulation during sensitization is more complex and diverse. The neuronal circuits mediating defensive reflexes contain a number of interneurons that respond to 5-HT in ways opposite to those of the SNs, showing a decrease in excitability and/or synaptic depression. Moreover, in addition to acting through a cAMP-PKA pathway within SNs, 5-HT is also capable of activating a variety of other protein kinases such as protein kinase C, extracellular signal-regulated kinases, and tyrosine kinases. This diversity of 5-HT responses during sensitization suggests the presence of multiple 5-HT receptor subtypes within the Aplysia central nervous system. Four 5-HT receptors have been cloned and characterized to date. Although several others probably remain to be characterized in molecular terms, especially the Gs-coupled 5-HT receptor capable of activating cAMP-PKA pathways, the multiplicity of serotonergic mechanisms recruited into action during learning in Aplysia can now be addressed from a molecular point of view.     

Quantitation of contacts among sensory, motor, and serotonergic neurons in the pedal ganglion of aplysia

Present models of long-term sensitization in Aplysia californica indicate that the enhanced behavioral response is due, at least in part, to outgrowth of sensory neurons mediating defensive withdrawal reflexes. Presumably, this outgrowth strengthens pre-existing connections by formation of new synapses with follower neurons. However, the relationship between the number of sensorimotor contacts and the physiological strength of the connection has never been examined in intact ganglia. As a first step in addressing this issue, we used confocal microscopy to examine sites of contact between sensory and motor neurons in naive animals. Our results revealed relatively few contacts between physiologically connected cells. In addition, the number of contact sites was proportional to the amplitude of the EPSP elicited in the follower motor neuron by direct stimulation of the sensory neuron. This is the first time such a correlation has been observed in the central nervous system. Serotonin is the neurotransmitter most closely examined for its role in modulating synaptic strength at the sensorimotor synapse. However, the structural relationship of serotonergic processes and sensorimotor synapses has never been examined. Surprisingly, serotonergic processes usually made contact with sensory and motor neurons at sites located relatively distant from the sensorimotor synapse. This result implies that heterosynaptic regulation is due to nondirected release of serotonin into the neuropil.     

In vitro analog of classical conditioning of feeding behavior in aplysia

The feeding behavior of Aplysia californica can be classically conditioned using tactile stimulation of the lips as a conditioned stimulus (CS) and food as an unconditioned stimulus (US). Moreover, several neural correlates of classical conditioning have been identified. The present study extended previous work by developing an in vitro analog of classical conditioning and by investigating pairing-specific changes in neuronal and synaptic properties. The preparation consisted of the isolated cerebral and buccal ganglia. Electrical stimulation of a lip nerve (AT4) and a branch of the esophageal nerve (En2) served as the CS and US, respectively. Three protocols were used: paired, unpaired, and US alone. Only the paired protocol produced a significant increase in CS-evoked fictive feeding. At the cellular level, classical conditioning enhanced the magnitude of the CS-evoked synaptic input to pattern-initiating neuron B31/32. In addition, paired training enhanced both the magnitude of the CS-evoked synaptic input and the CS-evoked spike activity in command-like neuron CBI-2. The in vitro analog of classical conditioning reproduced all of the cellular changes that previously were identified following behavioral conditioning and has led to the identification of several new learning-related neural changes. In addition, the pairing-specific enhancement of the CS response in CBI-2 indicates that some aspects of associative plasticity may occur at the level of the cerebral sensory neurons.     

Inflammation Causes a Long-Term Hyperexcitability in the Nociceptive Sensory Neurons of Aplysia

Nerve injury, tissue damage, and inflammation all cause hyperalgesia. A factor contributing to this increased sensitivity is a long-term (>24 hr) hyperexcitability (LTH) in the sensory neurons that mediate the responses. Using the cluster of nociceptive sensory neurons in Aplysia californica as a model, we are examining how inflammation induces LTH. A general inflammatory response was induced by inserting a gauze pad into the animal. Within 4 days, the gauze is enmeshed in an amorphous material that contains hemocytes, which comprise a cellular immune system. Concurrently, LTH appears in both ipsilateral and contralateral sensory neurons. The LTH is manifest as increased action potential discharge to a normalized stimulus. Immunocytochemistry revealed that hemocytes have antigens recognized by antibodies to TGFβ1, IL-6, and 5HT. When a localized inflammation was elicited on a nerve, hemocytes containing the TGFβ1 antigen were present near axons within the nerve and those containing the IL-6 were on the surface. Western blots of hemocytes, or of gauze that had induced a foreign body response, contained a 28-kD polypeptide recognized by the anti-TGFβ1 antibody. Exposure of the nervous system to recombinant human TGFβ1 elicited increased firing of the nociceptive neurons and a decrease in threshold. The TGFβ1 also caused an activation of protein kinase C (PKC) in axons but did not affect a kinase that is activated in axons after injury. Our findings, in conjunction with previous results, indicate that a TGFβ1-homolog can modulate the activity of neurons that respond to noxious stimuli. This system could also contribute to interactions between the immune and nervous systems via regulation of PKC.     

How do memory systems interact? Evidence from human classification learning

Studies of human classification learning using functional neuroimaging have suggested that basal ganglia and medial temporal lobe memory systems may interact during learning. We review these results and outline a set of possible mechanisms for such interactions. Effective connectivity analyses suggest that interaction between basal ganglia and medial temporal lobe are mediated by prefrontal cortex rather than by direct connectivity between regions. A review of possible neurobiological mechanisms suggests that interactions may be driven by neuromodulatory systems in addition to mediation by interaction of inputs to prefrontal cortical neurons. These results suggest that memory system interactions may reflect multiple mechanisms that combine to optimize behavior based on experience.     

Identification of a reinforcement pathway necessary for operant conditioning of head waving in Aplysia californica

The marine mollusc Aplysia californica exhibits a wide range of nonassociative and associative forms of learning. Recently, we found that the learning repertoire of Aplysia includes operant conditioning (Cook & Carew, 1986, 1989b). The behavior we examined is a naturally occurring, side-to-side head-waving response used by Aplysia in seeking food, obtaining a foothold, and egg laying. Aplysia can be operantly conditioned to reduce head-waving to one side of their body if such a response results in exposure to bright uniform-field illumination, which the animals find aversive. An essential step toward achieving a mechanistic understanding of operant conditioning is to identify and characterize the reinforcement pathway used during the learning. Toward this end, we wished to determine which of the peripheral visual pathways in Aplysia are critical for performance of the operant task. Previous experiments indicated that photic input from the optic and rhinophore nerves functionally inhibited motor neurons that participate in the operant response (head-waving), while photic input from the oral veil nerves excited these same motor neurons (Cook & Carew, 1989c). These findings suggested the hypothesis that one or both of these pathways could play an important role in mediating reinforcement during training. To explore this possibility we operantly trained animals that had received chronic bilateral transections of either the optic and rhinophore nerves or the oral veil nerves C1-C3 (in conjunction with transection of the optic and rhinophore nerves). We found that operant conditioning was not disrupted by ablation of input from the eyes and rhinophores. By contrast, ablation of input from the oral veil (together with that from the eyes and rhinophores) abolished operant conditioning. Thus, the oral veil nerves play a critical modulatory role in operant conditioning of head-waving. This observation further suggested that photic input from the oral veil is conveyed to the CNS via the oral veil nerves. In a final experiment we confirmed that stimulation of the oral veil with light evokes increased afferent activity in the oral veil nerves C1-C2. These results support the idea that the oral veil nerves contain processes that are critical components of the reinforcement pathway for operant conditioning of head-waving.     

Desensitization of postsynaptic glutamate receptors contributes to high-frequency homosynaptic depression of aplysia sensorimotor connections

Withdrawal reflexes of Aplysia are mediated in part by a monosynaptic circuit of sensory (SN) and motor (MN) neurons. A brief high-frequency burst of spikes in the SN produces excitatory postsynaptic potentials (EPSPs) that rapidly decrease in amplitude during the burst of activity. It is generally believed that this and other (i.e., low-frequency) forms of homosynaptic depression are entirely caused by presynaptic mechanisms (e.g., depletion of releasable transmitter). The present study examines the contribution that desensitization of postsynaptic glutamate receptors makes to homosynaptic depression. Bath application of cyclothiazide, an agent that reduces desensitization of non-NMDA glutamate receptors, reduced high-, but not low-frequency synaptic depression. Thus, a postsynaptic mechanism, desensitization of glutamate receptors, can also contribute to homosynaptic depression of sensorimotor synapses.     

Analysis of sequence-dependent interactions between transient calcium and transmitter stimuli in activating adenylyl cyclase in Aplysia: possible contribution to CS--US sequence requirement during conditioning

An important recent insight in a number of neurobiological systems is that during learning, individual dually regulated proteins with associative properties function as critical sites of stimulus convergence. During conditioning in Aplysia, the Ca2+ /calmodulin-sensitive adenylyl cyclase (AC) in mechanosensory neurons serves as a molecular site of interaction between Ca2+ and serotonin [5-hydroxytryptamine (5-HT)]-two signals that represent the CS and US in these cells. Conditioning requires that the CS and US be paired within a narrow time window and in the appropriate sequence. AC shows an analogous sequence preference: It is more effectively activated when a pulse of Ca2+ precedes a pulse of 5-HT than when the 5-HT precedes Ca2+. One mechanism that contributes to this sequence preference is that Ca2+/calmodulin binding to AC accelerates the rate of AC activation by receptor-Gs. We have identified two additional properties of AC activation that would cause pairing with Ca2+ preceding 5-HT to be more effective than simultaneous pairing or pairing with the reciprocal sequence: (1) Activation of Aplysia AC by a Ca2+ pulse rose with a delay compared with activation by a 5-HT pulse. (2) A late pulse of Ca2+, which arrived after 5-HT, acted, via calmodulin, to accelerate the decay of AC activation by receptor-Gs. Together, these activation properties of AC may contribute to the CS-US sequence requirement of classical conditioning.     

Inhibition of return interacts with the Simon effect: an omnibus analysis and its implications

Previous research has reported that the Simon effect (a type of stimulus-response [S-R] compatibility effect) and the inhibition of return effect (IOR; a late cuing effect) do not interact. In this brief report, we analyzed published and unpublished experiments that have examined these effects and found that IOR actually increases the Simon effect. This is a remarkable finding because most factors that delay reaction times (as IOR does) actually decrease the Simon effect. We examine this interaction within the context of seven interpretations of the effect that IOR may have on the task-irrelevant S-R code and two interpretations of the effect that IOR may have on the task-relevant S-R code, two components that underlie the Simon effect. The results falsified more than half of these interpretations, thus permitting future investigations to further reduce the number of theoretical alternatives.     

Dishabituation in Aplysia can involve either reversal of habituation or superimposed sensitization

Dishabituation has been thought to be due either to reversal of the process of habituation or to a second process equivalent to sensitization superimposed on habituation. One way to address this question is by testing whether dishabituation and sensitization can be dissociated. Previous studies using this approach in Aplysia have come to different conclusions about the nature of dishabituation, perhaps because those studies differed in many respects, including (1) whether they also observed transient behavioral inhibition, and (2) whether they used test stimuli that activated the LE siphon sensory neurons or as yet unidentified sensory neurons. To attempt to resolve the apparent contradictions between the previous studies, we have explored the importance of these two factors by performing a parametric study of dishabituation and sensitization of gill withdrawal in a simplified preparation that does not exhibit transient behavioral inhibition, using two different test stimuli that are known to activate the LE (Touch) or unidentified (Not Touch) sensory neurons. We find that dishabituation and sensitization in this preparation have similar time courses and generally similar functions of shock intensity. However, under one condition, with the Not Touch stimulus 2.5 min after the shock, dishabituation has a reverse effect of shock intensity. Additional analyses suggest that dishabituation with the Not Touch stimulus 2.5 min after the shock is due to reversal of habituation, whereas 12.5 min after the shock, dishabituation is due to superimposed sensitization. These results thus suggest that dishabituation may involve either process in the same preparation, and begin to define the conditions that favor one or the other.     

A presynaptic role for FMRP during protein synthesis-dependent long-term plasticity in Aplysia

Loss of the Fragile X mental retardation protein (FMRP) is associated with presumed postsynaptic deficits in mouse models of Fragile X syndrome. However, the possible presynaptic roles of FMRP in learning-related plasticity have received little attention. As a result, the mechanisms whereby FMRP influences synaptic function remain poorly understood. To investigate the cellular locus of the effects of FMRP on synaptic plasticity, we cloned the Aplysia homolog of FMRP and find it to be highly expressed in neurons. By selectively down-regulating FMRP in individual Aplysia neurons at the sensory-to-motor neuron synapse reconstituted in co-cultures, we demonstrate that FMRP functions both pre- and postsynaptically to constrain the expression of long-term synaptic depression induced by repeated pulses of FMRF-amide. In contrast, FMRP has little to no effect on long-term synaptic facilitation induced by repeated pulses of serotonin. Since other components of signaling pathways involved in plasticity appear to be conserved between Aplysia and mammalian neurons, our findings suggest that FMRP can participate in both pre- and postsynaptic regulation of enduring synaptic plasticity that underlies the storage of certain types of long-term memory.     

The Development of Attribute Dominance in the Knowledge Base

Two cuing, free-recall studies were conducted to test Bach and Underwood's (1970) hypothesis that acoustic encoding is dominant among second graders and semantic encoding is dominant among sixth graders. When retrieval cues were presented with to-be-remembered items at both input and output (Experiment 1), and when cues were presented only at output (Experiment 2), semantic cues were more efficient in elevating recall than were acoustic cues for both second and sixth graders. When these and other results generally found using recognition, sorting, incidental learning, and free-recall experimental designs are compared, it seems plausible that item presentation and memory-testing formats interact with age, and that these factors account for the different patterns of attribute dominance found in the literature. The knowledge base cannot be understood by focusing on either subject or task analyses, but only by focusing on interactions between subject and task variables as they change over time. The educational implications for young grade-school children are discussed.     

The development of attribute dominance in the knowledge base

Two cuing, free-recall studies were conducted to test Bach and Underwood's (1970) hypothesis that acoustic encoding is dominant among second graders and semantic encoding is dominant among sixth graders. When retrieval cues were presented with to-be-remembered items at both input and output (Experiment 1), and when cues were presented only at output (Experiment 2), semantic cues were more efficient in elevating recall than were acoustic cues for both second and sixth graders. When these and other results generally found using recognition, sorting, incidental learning, and free-recall experimental designs are compared, it seems plausible that item presentation and memory-testing formats interact with age, and that these factors account for the different patterns of attribute dominance found in the literature. The knowledge base cannot be understood by focusing on either subject or task analyses, but only by focusing on interactions between subject and task variables as they change over time. The educational implications for young grade-school children are discussed.     

Social tuning of the self: consequences for the self-evaluations of stereotype targets

These experiments examined how social interactions with individuals who ostensibly have stereotype-relevant views affect the self-evaluations of stereotype targets. Participants believed they were going to interact, or actually interacted, with a person who ostensibly had stereotype-consistent or stereotype-inconsistent views about their social group. Consistent with shared reality theory, participants' self-evaluations (Experiments 1, 2, and 3) and behavior (Experiment 2) corresponded with the ostensible views of the other person when affiliative motivation was high. This occurred even when it was likely to be detrimental to participants' nonaffiliative outcomes (Experiment 3). Experiment 4 showed that self-evaluative shift away from the ostensible views of another person was a function of social distance motives, also consistent with shared reality theory.