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.

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.

Long-term sensitization training primes Aplysia for further learning.

Repetitive, unilateral stimulation of Aplysia induces long-term sensitization (LTS) of ipsilaterally elicited siphon-withdrawal responses. Whereas some morphological effects of training appear only on ipsilateral sensory neurons, others appear bilaterally. We tested the possibility that contralateral morphological modifications may have functional significance. Therefore, we examined whether LTS training primes subsequent sensitization. Twenty-four hours after LTS training the effects of brief shock treatment (BST) were examined. BST failed to sensitize animals that had previously received either 4-d control treatment or 4-d ipsilateral LTS training. In contrast, BST did sensitize animals that had previously received 4-d contralateral LTS training, suggesting the presence of a latent trace that primes the animal for further learning.

Dissociation of morphological and physiological changes associated with long-term memory in aplysia.

Neurite outgrowth is a process commonly thought to contribute to long-term learning by formation of new synaptic contacts. The behavioral effects of long-term sensitization training in Aplysia were restricted to the trained side of the animal as were changes in strength of the sensorimotor synapse. In contrast, training produced varicosity formation on both sides of the animal. Appositions with follower neurons, however, were enhanced only on the trained side. The dissociation of structural and functional correlates suggests that key regulatory steps are downstream from outgrowth, possibly in the targeting of new processes and activation of new synapses.

Localized neuronal outgrowth induced by long-term sensitization training in aplysia.

Biophysical, biochemical, and morphological studies have implicated sensory neurons as key sites of plasticity in the formation and retention of the memory of long-term sensitization in Aplysia californica. This study examined the effects of different sensitization training protocols on the structure of sensory neurons mediating the tail-siphon withdrawal reflex. A 4 d training period produced a robust localized outgrowth in these sensory neurons observed 24 hr after the end of training. These changes are consistent with previous results in siphon sensory neurons (Bailey and Chen, 1988a). In contrast, 1 d of sensitization training, which has been shown to effectively induce long-term behavioral sensitization and synaptic facilitation (Frost et al., 1985; Cleary et al., 1998), is not associated with morphological changes in tail sensory neurons at either 24 hr or 4 d after training. Similarly, a single treatment with the growth factor TGF-beta, which also induced facilitation, did not alter sensory neuron morphology. The different effectiveness of the two protocols was not simply a reflection of the number of stimuli presented, because a 1 d massed training protocol did not produce sensitization 24 hr after training, nor did it induce neuronal outgrowth. These results suggest that extensive sensitization training is required to induce neuronal outgrowth in tail sensory neurons, indicating that the memory of long-term sensitization induced by 1 d of training is mechanistically different from that induced by 4 d of training. Moreover, the induction of a form of long-term sensitization associated with neuronal outgrowth does not appear to be a function of the amount of stimulation but does appear to be dependent on the temporal spacing of the stimulation over multiple days.