Role of serotonin in the lack of sensitization caused by prolonged food deprivation in Aplysia.

Food deprivation may cause neurological dysfunctions including memory impairment. The mollusk Aplysia is a suitable animal model to study prolonged food deprivation-induced memory deficits because it can sustain up to 14 days of food deprivation (14DFD). Sensitization of defensive withdrawal reflexes has been used to illustrate the detrimental effects of 14DFD on memory formation. Under normal feeding conditions (i.e., two days food deprivation, 2DFD), aversive stimuli lead to serotonin (5-HT) release into the hemolymph and neuropil, which mediates sensitization and its cellular correlates including increased excitability of tail sensory neurons (TSNs). Recent studies found that 14DFD prevents both short-term and long-term sensitization, as well as short-term increased excitability of TSNs induced by in vitro aversive training. This study investigated the role of 5-HT in the absence of sensitization and TSN increased excitability under 14DFD. Because 5-HT is synthesized from tryptophan obtained through diet, and its exogeneous application alone induces sensitization and increases TSN excitability, we hypothesized that 1) 5-HT level may be reduced by 14DFD and 2) 5-HT may still induce sensitization and TSN increased excitability in 14DFD animals. Results revealed that 14DFD significantly decreased hemolymph 5-HT level, which may contribute to the lack of sensitization and its cellular correlates, while ganglia 5-HT level was not changed. 5-HT exogenous application induced sensitization in 14DFD Aplysia, albeit smaller than that in 2DFD animals, suggesting that this treatment can only induce partial sensitization in food deprived animals. Under 14DFD, 5-HT increased TSN excitability indistinguishable from that observed under 2DFD. Taken together, these findings characterize 5-HT metabolic deficiency under 14DFD, which may be compensated, at least in part, by 5-HT exogenous application.

Using an invertebrate model to investigate the mechanisms of short-term memory deficits induced by food deprivation.

Although prolonged food deprivation is known to cause memory deficits, the underlying mechanisms are only partially understood. In this study, we began to investigate the cellular substrates of food deprivation-induced memory impairments in the invertebrate Aplysia. Following a single trial of noxious stimuli, Aplysia concurrently express short-term sensitization (an elementary form of learning in which withdrawal reflexes are enhanced) and short-term feeding suppression for at least 15 min. Cellular correlates of sensitization and feeding suppression include increased excitability of the tail sensory neurons (TSNs) controlling the withdrawal reflexes, and decreased excitability of feeding decision-making neuron B51, respectively. Recently, 14 days of food deprivation (14DFD) was reported to break the co-expression of sensitization and feeding suppression in Aplysia without health deterioration. Specifically, under 14DFD, sensitization was completely prevented while feeding suppression was present albeit attenuated. This study explored the cellular mechanisms underlying the absent sensitization and reduced feeding suppression under 14DFD. A reduced preparation was used to evaluate the short-term cellular modifications induced by delivering an aversive training protocol in vitro. TSN excitability failed to increase following in vitro training under 14DFD, suggesting that the lack of sensitization may be a consequence of the fact that TSN excitability failed to increase. B51 excitability also failed to decrease following in vitro training, indicating that additional neurons may contribute to the conserved albeit reduced feeding suppression in 14DFD animals. This study lays the foundations for the future use of the Aplysia model system to investigate the mechanisms underlying the memory impairments induced by prolonged food deprivation.

Critical role of protein kinase G in the long-term balance between defensive and appetitive behaviors induced by aversive stimuli in Aplysia.

This study investigated the signaling cascades involved in the long-term storage of the balance between defensive and appetitive behaviors observed when the mollusk Aplysia is exposed to aversive experience. In Aplysia, repeated trials of aversive stimuli induce concurrent sensitization of defensive withdrawal reflexes and suppression of feeding for at least 24 h. This long-term storage of the balance between withdrawal reflexes and feeding is sustained, at least in part, by increased excitability of the tail sensory neurons (SNs) controlling the withdrawal reflexes, and by decreased excitability of feeding decision-making neuron B51. Nitric oxide (NO) is required for the induction of both long-term sensitization and feeding suppression. At the cellular level, NO is also required for long-term decreased B51 excitability but not for long-term increased SN excitability. Here, we characterized the signaling cascade downstream of NO contributing to the long-term storage of the balance between withdrawal reflexes and feeding. We found protein kinase G (PKG) necessary for both long-term sensitization and feeding suppression, indicating that a NO-PKG cascade governs the long-term storage of the balance between defensive and appetitive responses in Aplysia. The role of PKG on feeding suppression was paralleled at the cellular level where a cGMP-PKG pathway was required for long-term decreased B51 excitability. In the defensive circuit, the cGMP-PKG pathway was not necessary for long-term increased SN excitability, suggesting that other cellular correlates of long-term sensitization might depend on the GMP-PKG cascade to sustain the behavioral change.

Role of nitric oxide in the induction of the behavioral and cellular changes produced by a common aversive stimulus in Aplysia.

Although it is well documented that exposure to aversive stimuli induces modulation of neural circuits and subsequent behavioral changes, the means by which an aversive stimulus concomitantly alters behaviors of different natures (e.g., defensive and appetitive) remains unclear. Here, we addressed this issue by using the learning-induced concurrent modulation of defensive and appetitive behaviors that occurs when the mollusk Aplysia is exposed to aversive stimuli. In Aplysia, aversive stimuli concomitantly enhance withdrawal reflexes (i.e., sensitization) and suppress feeding. Sensitization and feeding suppression, which are expressed in the short term and long term, depending on the training protocol, are accompanied by increased excitability of the tail sensory neurons (TSNs) controlling the withdrawal reflexes, and by decreased excitability of feeding decision-making neuron B51, respectively. Serotonin (5-HT) has been shown to mediate sensitization, but not feeding suppression. In this study, we examined which other neurotransmitter might be responsible for feeding suppression and its underlying cellular changes. Our results indicate that nitric oxide (NO) contributes to both short-term and long-term feeding suppression, as well as to the underlying decreased B51 excitability. NO was also necessary for the induction of long-term sensitization and for the expression of short-term increased TSN excitability in vitro, revealing a previously undocumented interaction between 5-HT and NO signaling cascades in sensitization. Overall, these results revealed a scenario in which multiple modulators contribute to the widespread changes induced by sensitizing stimuli in Aplysia.

cGMP mediates short- and long-term modulation of excitability in a decision-making neuron in Aplysia.

In elementary neural circuits, changes in excitability can have a strong impact in the expression of a given behavior. One example is provided by B51, a neuron with decision-making properties in the feeding neural circuit of the mollusk Aplysia. The excitability of B51 is bidirectionally modulated by external and internal stimuli in a manner that is consistent with the corresponding induced changes in feeding behavior. For example, in operant reward learning, which up-regulates feeding, B51 excitability is increased via a cAMP-dependent mechanism. Conversely, following training protocols with aversive stimuli, which down-regulate feeding, B51 excitability is decreased. In this study, we tested the hypothesis that B51 decreased excitability may be mediated by another cyclic nucleotide, cGMP. Our results revealed that iontophoretic injection of cGMP was capable of inducing both short-term (45 min) and long-term (24 h) reduction of B51 excitability. We next investigated which biochemical trigger could increase cGMP cytosolic levels. The neurotransmitter nitric oxide was found to decrease B51 excitability through the activation of the soluble guanylyl cyclase. These findings indicate that a cGMP-dependent pathway modulates B51 excitability in a manner opposite of cAMP, indicating that distinct cyclic-nucleotide pathways bidirectionally regulate the excitability of a decision-making neuron.

Effects of internal and external factors on the budgeting between defensive and non-defensive responses in Aplysia.

Following exposure to aversive stimuli, organisms budget their behaviors by augmenting defensive responses and reducing/suppressing non-defensive behaviors. This budgeting process must be flexible to accommodate modifications in the animal's internal and/or external state that require the normal balance between defensive and non-defensive behaviors to be adjusted. When exposed to aversive stimuli, the mollusk Aplysia budgets its behaviors by concurrently enhancing defensive withdrawal reflexes (an elementary form of learning known as sensitization) and suppressing feeding. Sensitization and feeding suppression are consistently co-expressed following different training protocols and share common temporal domains, suggesting that they are interlocked. In this study, we attempted to uncouple the co-expression of sensitization and feeding suppression using: 1) manipulation of the animal's motivational state through prolonged food deprivation and 2) extended training with aversive stimuli that induces sensitization lasting for weeks. Both manipulations uncoupled the co-expression of the above behavioral changes. Prolonged food deprivation prevented the expression of sensitization, but not of feeding suppression. Following the extended training, sensitization and feeding suppression were co-expressed only for a limited time (i.e., 24 h), after which feeding returned to baseline levels as sensitization persisted for up to seven days. These findings indicate that sensitization and feeding suppression are not interlocked and that their co-expression can be uncoupled by internal (prolonged food deprivation) and external (extended aversive training) factors. The different strategies, by which the co-expression of sensitization and feeding suppression was altered, provide an example of how budgeting strategies triggered by an identical aversive experience can vary depending on the state of the organism.

A novel in vitro analog expressing learning-induced cellular correlates in distinct neural circuits.

When presented with noxious stimuli, Aplysia exhibits concurrent sensitization of defensive responses, such as the tail-induced siphon withdrawal reflex (TSWR) and suppression of feeding. At the cellular level, sensitization of the TSWR is accompanied by an increase in the excitability of the tail sensory neurons (TSNs) that elicit the reflex, whereas feeding suppression is accompanied by decreased excitability of B51, a decision-making neuron in the feeding neural circuit. The goal of this study was to develop an in vitro analog coexpressing the above cellular correlates. We used a reduced preparation consisting of buccal, cerebral, and pleural-pedal ganglia, which contain the neural circuits controlling feeding and the TSWR, respectively. Sensitizing stimuli were delivered in vitro by electrical stimulation of afferent nerves. When trained with sensitizing stimuli, the in vitro analog expressed concomitant increased excitability in TSNs and decreased excitability in B51, which are consistent with the occurrence of sensitization and feeding suppression induced by in vivo training. This in vitro analog expressed both short-term (15 min) and long-term (24 h) excitability changes in TSNs and B51, depending on the amount of training administered. Finally, in vitro application of serotonin increased TSN excitability without altering B51 excitability, mirroring the in vivo application of the monoamine that induces sensitization, but not feeding suppression.

Long-term sensitization training in Aplysia decreases the excitability of a decision-making neuron through a sodium-dependent mechanism.

In Aplysia, long-term sensitization (LTS) occurs concurrently with a suppression of feeding. At the cellular level, the suppression of feeding is accompanied by decreased excitability of decision-making neuron B51. We examined the contribution of voltage-gated Na+ and K+ channels to B51 decreased excitability. In a pharmacologically isolated Na+ channels environment, LTS training significantly increased B51 firing threshold, compared with untrained controls. Conversely, in a pharmacologically isolated K+ channels environment, no differences were observed between trained and untrained animals in either amplitude or area of B51 K+-dependent depolarizations. These findings suggest that Na+ channels contribute to the decrease in B51 excitability induced by LTS training.

Change in excitability of a putative decision-making neuron in Aplysia serves as a mechanism in the decision not to feed following food satiation.

Although decision making is a ubiquitous function, the understanding of its underlying mechanisms remains limited, particularly at the single-cell level. In this study, we used the decision not to feed that follows satiation in the marine mollusk Aplysia to examine the role of putative decision-making neuron B51 in this process. B51 is a neuron in the feeding neural circuit that exhibits decision-making characteristics in vitro, which bias the circuit toward producing the motor programs responsible for biting behavior. Once satiated, Aplysia decided not to bite for a prolonged period of time (≥24h) when presented with a food stimulus that normally elicits feeding in non-satiated animals. Twenty-four hours after satiation, suppressed feeding was accompanied by a significant decrease of B51 excitability compared to the control group of unfed animals. No differences were measured in B51 resting membrane properties or synaptic input to B51 between the satiated and control groups. When B51 properties were measured at a time point in which feeding had recovered from the suppressive effects of satiation (i.e., 96 h after satiation), no difference in B51 excitability was observed between satiated and control groups. These findings indicate that B51 excitability changes in a manner that is coherent with the modifications in biting resulting from food satiation, thus implicating this neuron as a site of plasticity underlying the decision not to bite following food satiation in Aplysia.

Effects of aversive stimuli beyond defensive neural circuits: reduced excitability in an identified neuron critical for feeding in Aplysia.

In Aplysia, repeated trials of aversive stimuli produce long-term sensitization (LTS) of defensive reflexes and suppression of feeding. Whereas the cellular underpinnings of LTS have been characterized, the mechanisms of feeding suppression remained unknown. Here, we report that LTS training induced a long-term decrease in the excitability of B51 (a decision-making neuron in the feeding circuit) that recovered at a time point in which LTS is no longer observed (72 h post-treatment). These findings indicate B51 as a locus of plasticity underlying feeding suppression. Finally, treatment with serotonin to induce LTS failed to alter feeding and B51 excitability, suggesting that serotonin does not mediate the effects of LTS training on the feeding circuit.

Rapid and persistent suppression of feeding behavior induced by sensitization training in Aplysia.

In Aplysia, noxious stimuli induce sensitization of defensive responses. However, it remains largely unknown whether such stimuli also alter nondefensive behaviors. In this study, we examined the effects of noxious stimuli on feeding. Strong electric shocks, capable of inducing sensitization, also led to the suppression of feeding. The use of multiple training protocols revealed that the time course of the suppression of feeding was analogous to that of sensitization. In addition, the suppression of feeding was present only at the time points in which sensitization was expressed. These results suggest that, in Aplysia, noxious stimuli may produce concurrent changes in neural circuits controlling both defensive and nondefensive behaviors.

Molecular mechanisms of short-term habituation in the leech Hirudo medicinalis.

Although habituation is ubiquitous in the animal kingdom, its underlying mechanisms remain poorly understood. In this study, we began to explore the molecular cascades underlying short-term habituation in the leech Hirudo medicinalis. In H. medicinalis, a training paradigm, consisting of low-frequency repetitive electrical stimulation of the skin, produces a gradual increase in the latency to swim that spontaneously recovers within 20-30 min. As first step in determining the molecular pathways in short-term habituation, we examined the role of Ca(2+). Both Ca(2+) influx through voltage-gated channels and Ca(2+) release from intracellular stores were found to contribute to short-term habituation. The analysis of the downstream targets of elevated cytosolic Ca(2+) revealed that the activation of the phosholipase A(2) was required for the induction of short-term habituation. Finally, we reported that the recruitment of arachidonic acid metabolites, generated by the 5-lipoxygenase pathway, was also necessary for the induction of swim induction habituation. These results provide the framework for a comprehensive characterization of the molecular underpinnings of habituation. This outcome will allow us to compare the mechanisms of habituation with those underlying other forms of nonassociative learning in the leech, such as sensitization and dishabituation, and, more in general, with those governing habituation in different vertebrate and invertebrate model systems.

More than synaptic plasticity: role of nonsynaptic plasticity in learning and memory.

Decades of research on the cellular mechanisms of memory have led to the widely held view that memories are stored as modifications of synaptic strength. These changes involve presynaptic processes, such as direct modulation of the release machinery, or postsynaptic processes, such as modulation of receptor properties. Parallel studies have revealed that memories might also be stored by nonsynaptic processes, such as modulation of voltage-dependent membrane conductances, which are expressed as changes in neuronal excitability. Although in some cases nonsynaptic changes can function as part of the engram itself, they might also serve as mechanisms through which a neural circuit is set to a permissive state to facilitate synaptic modifications that are necessary for memory storage.

Changes in neuronal excitability serve as a mechanism of long-term memory for operant conditioning.

Learning can lead to changes in the intrinsic excitability of neurons. However, the extent to which these changes persist and the role they have in the expression of memory remain unclear. We found that in vitro analogs of operant conditioning produced a long-term (24 h) increase in the excitability of an identified neuron (B51) that is critical for the expression of feeding in Aplysia. This increase in excitability, which was cAMP dependent, contributed to the associative modification of the feeding circuitry, providing a mechanism for long-term memory.

Classical and operant conditioning differentially modify the intrinsic properties of an identified neuron.

A long-standing debate in neuroscience is whether classical and operant conditioning are mechanistically similar or distinct. The feeding behavior of Aplysia provides a model system suitable for addressing this question. Here we report that classical and operant conditioning of feeding behavior differentially modify the intrinsic excitability of neuron B51, a critical element for the expression of the feeding response, thus revealing that these two forms of associative learning differ at the cellular level.

Reinforcement in an in vitro analog of appetitive classical conditioning of feeding behavior in Aplysia: blockade by a dopamine antagonist.

In a recently developed in vitro analog of appetitive classical conditioning of feeding in Aplysia, the unconditioned stimulus (US) was electrical stimulation of the esophageal nerve (En). This nerve is rich in dopamine (DA)-containing processes, which suggests that DA mediates reinforcement during appetitive conditioning. To test this possibility, methylergonovine was used to antagonize DA receptors. Methylergonovine (1 nM) blocked the pairing-specific increase in fictive feeding that is usually induced by in vitro classical conditioning. The present results and previous observation that methylergonovine also blocks the effects of contingent reinforcement in an in vitro analog of appetitive operant conditioning suggest that DA mediates reinforcement for appetitive associative conditioning of feeding in Aplysia.

Role for calcium signaling and arachidonic acid metabolites in the activity-dependent increase of AHP amplitude in leech T sensory neurons.

Previous studies have revealed a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) neurons of the leech Hirudo medicinalis. The firing of T cells is characterized by an AHP, which is mainly due to the activity of the Na+/K+ ATPase. Low-frequency repetitive stimulation of T neurons leads to a robust increment of the AHP amplitude, which is correlated with a synaptic depression between T neuron and follower cells. In the present study, we explored the molecular cascades underlying the AHP increase. We tested the hypothesis that this activity-dependent phenomenon was triggered by calcium influx during neural activity by applying blockers of voltage-dependent Ca2+ channels. We report that AHP increase requires calcium influx that, in turn, induces release of calcium from intracellular stores so sustaining the enhancement of AHP. An elevation of the intracellular calcium can activate the cytosolic isoforms of the phosholipase A2 (PLA2). Therefore we analyzed the role of PLA2 in the increase of the AHP, and we provide evidence that not only PLA2 but also the recruitment of arachidonic acid metabolites generated by the 5-lipoxygenase pathway are necessary for the induction of AHP increase. These data indicate that a sophisticated cascade of intracellular signals links the repetitive discharge of T neurons to the activation of molecular pathways, which finally may alter the activity of critical enzymes such as the Na+/K+ ATPase, that sustains the generation of the AHP and its increase during repetitive stimulation. These results also suggest the potential importance of the poorly studied 5-lipoxygenase pathway in forms of neuronal plasticity.

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.

Activity-dependent increase of the AHP amplitude in T sensory neurons of the leech.

We identified a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) sensory neurons of the leech Hirudo medicinalis. Repetitive intracellular stimulation with 30 trains of depolarizing impulses at 15-s inter-stimulus interval (ISI) led to an increase of the AHP amplitude (~60% of the control). The enhancement of AHP lasted for >/=15 min. The AHP increase was also elicited when a T neuron was activated by repetitive stimulation of its receptive field. The ISI was a critical parameter for the induction and maintenance of AHP enhancement. ISI duration had to fit within a time window with the upper limit of 20 s to make the training effective to induce an enhancement of the AHP amplitude. After recovery from potentiation, AHP amplitude could be enhanced once again by delivering another training session. The increase of AHP amplitude persisted in high Mg(2+) saline, suggesting an intrinsic cellular mechanism for its induction. Previous investigations reported that AHP of leech T neurons was mainly due to the activity of the Na(+)/K(+) ATPase and to a Ca(2+)-dependent K(+) current (I(K/Ca)). In addition, it has been demonstrated that serotonin (5HT) reduces AHP amplitude through the inhibition of the Na(+)/K(+) ATPase. By blocking the I(K/Ca) with pharmacological agents, such as cadmium and apamin, we still observed an increase of the AHP amplitude after repetitive stimulation, whereas 5HT application completely inhibited the AHP increment. These data indicate that the Na(+)/K(+) ATPase is involved in the induction and maintenance of the AHP increase after repetitive stimulation. Moreover, the AHP increase was affected by the level of serotonin in the CNS. Finally, the increase of the AHP amplitude produced a lasting depression of the synaptic connection between two T neurons, suggesting that this activity-dependent phenomenon might be involved in short-term plasticity associated with learning processes.