Intracellular injection of cAMP induces a long-term reduction of neuronal K+ currents.

Intracellular signals that trigger long-term (24-hour) changes in membrane currents in identified neurons of Aplysia have been examined in order to understand the cellular mechanisms underlying long-term sensitization. Adenosine 3',5'-monophosphate (cAMP) was directly injected into individual sensory neurons to mimic the effects of sensitization training at the single cell level. Potassium currents of these cells were reduced 24 hours after injection of cAMP; these currents were similar to those reduced 24 hours after behavioral sensitization. These results suggest that cAMP is part of the intracellular signal that induces long-term sensitization in Aplysia.

Long-term sensitization in Aplysia: biophysical correlates in tail sensory neurons.

A fundamental problem in the cellular analysis of learning and memory is the identification of the neuronal substrates of long-term information storage and their relation to short-term cellular alterations. In this report, biophysical correlates of long-term sensitization of a simple withdrawal reflex in the mollusc Aplysia were examined. A voltage-clamp analysis of the sensory neurons that control the reflex, 24 hours after sensitization training, revealed a significant reduction in net outward current. The results indicate that one mechanism for the storage of long-term sensitization is the regulation of membrane currents that influence the characteristics of the action potential and the excitability of individual neurons. The results also provide insights into the relation between short- and long-term sensitization in that the biophysical loci involved in the storage of long-term sensitization appear similar to those involved in short-term sensitization.

Associative conditioning of single sensory neurons suggests a cellular mechanism for learning.

A cellular analog of associative learning has been demonstrated in individual sensory neurons of the tail withdrawal reflex of Aplysia. Sensory cells activated by intracellular current injection shortly before a sensitizing shock to the animal's tail display significantly more facilitation of their monosynaptic connections to a tail motor neuron than cells trained either with intracellular stimulation unpaired to tail shock or with tail shock alone. This associative effect is acquired rapidly and is expressed as a temporally specific amplification of heterosynaptic facilitation. The results suggest that activity-dependent neuromodulation may be a mechanism underlying associative information storage and point to aspects of subcellular processes that might be involved in the formation of neural associations.

Long-term synaptic changes produced by a cellular analog of classical conditioning in Aplysia.

A change in synaptic strength arising from the activation of two neuronal pathways at approximately the same time is a form of associative plasticity and may underlie classical or Pavlovian conditioning. A cellular analog of a classical conditioning protocol produces short-term associative plasticity at the connections between sensory and motor neurons in Aplysia. A similar training protocol produced long-term (24-hour) enhancement of excitatory postsynaptic potentials (EPSPs). EPSPs produced by sensory neurons in which activity was paired with a reinforcing stimulus were significantly larger than unpaired controls 24 hours after training. Thus, associative plasticity at the sensory to motor neuron connection can occur in a long-term form in addition to the short-term form. In this system, it should be possible to analyze the molecular mechanisms underlying long-term associative plasticity and classical conditioning.

Role of transforming growth factor-beta in long-term synaptic facilitation in Aplysia.

The role of transforming growth factor-beta (TGF-beta) in long-term synaptic facilitation was examined in isolated Aplysia ganglia. Treatment with TGF-beta1 induced long-term facilitation (24 and 48 hours), but not short-term (5 to 15 minutes) or intermediate-term (2 to 4 hours) facilitation. The long-term effects of TGF-beta1 were not additive with those of serotonin. Moreover, serotonin-induced facilitation was blocked by an inhibitor of TGF-beta. Thus, activation of TGF-beta may be part of the cascade of events underlying long-term sensitization, consistent with the hypothesis that signaling molecules that participate in development also have roles in adult neuronal plasticity.

Ionic conductance mechanisms contributing to the electrophysiological properties of neurons.

Neurons have a multiplicity of ionic conductance mechanisms, the interactions of which determine in part the response of a neuron to chemical and electrical synaptic interactions, firing patterns, excitability, membrane potential and action-potential waveform. Several papers published in the past year have provided important new information on the role that ionic conductance mechanisms play in determining the electrophysiological properties of neurons, and how subtle differences can contribute to considerable variability of response.

Levels of serotonin in the hemolymph of Aplysia are modulated by light/dark cycles and sensitization training.

Serotonin (5-hydroxytryptamine, 5-HT) modulates the behavior and physiology of both vertebrate and invertebrate animals. Effects of injections of 5-HT and the morphology of the serotonergic system of Aplysia indicate that 5-HT may have a humoral, in addition to a neurotransmitter, role. To study possible humoral roles of 5-HT, we measured 5-HT in the hemolymph. The concentration of 5-HT in the hemolymph was approximately 18 nM, a value close to previously reported thresholds for eliciting physiological responses. The concentration of 5-HT in the hemolymph expressed a diurnal rhythm. In addition, electrical stimulation that leads to long-term sensitization significantly increased levels of 5-HT in the hemolymph during training, 1.5 hr after training, and 24 hr after training. Moreover, levels of 5-HT in the hemolymph were significantly correlated with the magnitude of sensitization. The half-life of an increase in 5-HT in the hemolymph was approximately 0.5 hr. Therefore, the persistent increase of 5-HT in the hemolymph 24 hr after sensitization training indicates that training caused a long-lasting increase in the release of 5-HT. This long-lasting increase in 5-HT in the hemolymph was blocked by treatment with an inhibitor of protein synthesis during training. Based on the levels of 5-HT in the hemolymph and its regulation by environmental events, we propose that 5-HT has a humoral role in regulation of the behavioral state of Aplysia. In support of this hypothesis, we found that increasing levels of 5-HT in the hemolymph led to significant alterations in feeding behavior. Increasing levels of 5-HT during the daytime when they were normally low increased the latency to assume feeding posture from daytime to nighttime values.

Modeling circadian oscillations with interlocking positive and negative feedback loops.

Both positive and negative feedback loops of transcriptional regulation have been proposed to be important for the generation of circadian rhythms. To test the sufficiency of the proposed mechanisms, two differential equation-based models were constructed to describe the Neurospora crassa and Drosophila melanogaster circadian oscillators. In the model of the Neurospora oscillator, FRQ suppresses frq transcription by binding to a complex of the transcriptional activators WC-1 and WC-2, thus yielding negative feedback. FRQ also activates synthesis of WC-1, which in turn activates frq transcription, yielding positive feedback. In the model of the Drosophila oscillator, PER and TIM are represented by a "lumped" variable, "PER." PER suppresses its own transcription by binding to the transcriptional regulator dCLOCK, thus yielding negative feedback. PER also binds to dCLOCK to de-repress dclock, and dCLOCK in turn activates per transcription, yielding positive feedback. Both models displayed circadian oscillations that were robust to parameter variations and to noise and that entrained to simulated light/dark cycles. Circadian oscillations were only obtained if time delays were included to represent processes not modeled in detail (e.g., transcription and translation). In both models, oscillations were preserved when positive feedback was removed.

Transforming growth factor beta1 alters synapsin distribution and modulates synaptic depression in Aplysia.

Transforming growth factor beta1 (TGF-beta1) induces long-term synaptic facilitation and long-term increases in excitability in Aplysia. Here we report that this growth factor has acute effects as well. Treatment of pleural-pedal ganglia with TGF-beta1 for 5 min activated mitogen-activated protein kinase (MAPK) and stimulated the phosphorylation of synapsin in a MAPK-dependent manner. This phosphorylation appeared to modulate synapsin distribution in cultured sensory neurons. Control neurons exhibited a punctate distribution of synapsin along neurites, which appeared to represent high concentration aggregates of synapsin. TGF-beta1-treated sensory neurons showed a significant reduction in the number of these puncta, an effect that was blocked by the MAP/ERK kinase inhibitor U0126. The functional consequence of TGF-beta1 was tested by examining its effects on synaptic transmission at the sensorimotor synapse. Application of TGF-beta1 reduced the magnitude of synaptic depression. This effect was dependent on MAPK, consistent with the hypothesis that TGF-1 mobilizes synaptic vesicles through the phosphorylation of synapsin.

Classical conditioning of feeding in Aplysia: I. Behavioral analysis.

A training protocol was developed to classically condition feeding behavior in Aplysia californica using tactile stimulation of the lips as the conditional stimulus (CS) and food as the unconditional stimulus (US). Paired training induced a greater increase in the number of bites to the CS than unpaired training or US-only stimulation. Memory for classical conditioning was retained for at least 24 hr. The organization of the reinforcement pathway that supports classical conditioning was analyzed in additional behavioral experiments. No evidence was found for the contribution to appetitive reinforcement of US-mediating pathways originating in the lips of the animals. Bilateral lesions of the anterior branch of the esophageal nerve, which innervates parts of the foregut, however, were found to attenuate classical conditioning. Thus, it appears likely that reinforcement during appetitive classical conditioning of feeding was mediated by afferent pathways that originate in the foregut. The companion paper () describes two neurophysiological correlates of the classical conditioning.

Classical conditioning of feeding in Aplysia: II. Neurophysiological correlates.

Feeding behavior in Aplysia californica can be classically conditioned using tactile stimulation of the lips as conditional stimulus (CS) and food as unconditional stimulus (US) [ (companion paper)]. Conditioning resulted in an increase in the number of CS-evoked bites that persisted for at least 24 hr after training. In this study, neurophysiological correlates of classical conditioning training were identified and characterized in an in vitro preparation of the cerebral and buccal ganglia. Stimulation of a lip nerve (AT(4)), which mediates mechanosensory information, resulted in a greater number of buccal motor patterns (BMPs) in ganglia isolated from animals that had received paired training than in ganglia from control animals. The majority of the evoked BMPs were classified as ingestion-like patterns. Intracellular recordings from pattern-initiating neuron B31/32 revealed that stimulation of AT(4) evoked greater excitatory input in B31/32 in preparations from animals that had received paired training than from control animals. In contrast, excitatory input to buccal neuron B4/5 in response to stimulation of AT(4) was not significantly increased by paired training. Moreover, correlates of classical conditioning were specific to stimulation of AT(4). The number of spontaneously occurring BMPs and the intrinsic properties of two buccal neurons (B4/5 and B31/32) did not differ between groups. These results suggest that appetitive classical conditioning of feeding resulted in the pairing-specific strengthening of the polysynaptic pathway between afferent fibers and pattern-initiating neurons of the buccal central pattern generator.

Serotoninergic varicosities make synaptic contacts with pleural sensory neurons of Aplysia.

Serotonin is a modulatory neurotransmitter that produces many of the cellular changes associated with sensitization of reflexes in Aplysia. These changes have been carefully documented in sensory neurons located in the abdominal ganglion that mediate the gill-siphon withdrawal reflex and in sensory neurons located in the pleural ganglion that mediate the tail-siphon withdrawal reflex. Although serotonin appears to be necessary for sensitization, there is no direct evidence that serotoninergic neurons make synaptic contacts with sensory neurons. In this study, the immunoperoxidase technique was used to label serotonin-immunoreactive neurites surrounding the cell bodies of sensory neurons in the pleural ganglion. Serotonin-immunoreactive neurites had varicosities whose mean short axis diameter was 1.1 +/- 0.6 microns (mean +/- S.D.). The shape of the size distribution was skewed toward larger sizes, however, suggesting that there were multiple subpopulations of varicosities. One subpopulation was that of varicosities located at branch points whose average short axis diameter was larger than normal (1.7 +/- 0.5 microns). Serotonin-immunoreactive varicosities were directly apposed to the sensory neurons without intervening glial cells. In most contacts, serotonin-immunoreactive neurites invaginated into the plasma membranes of the sensory neurons. There were also a few contacts onto spinelike processes, but these were flat rather than invaginated. Serotoninergic neurons whose activity produces changes in the electrophysiological properties of sensory neurons have been identified, but this study provides the first direct evidence for synaptic connections between serotoninergic neurons and sensory neurons in Aplysia.

Localization of glutamate and glutamate transporters in the sensory neurons of Aplysia.

The sensorimotor synapse of Aplysia has been used extensively to study the cellular and molecular basis for learning and memory. Recent physiologic studies suggest that glutamate may be the excitatory neurotransmitter used by the sensory neurons (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779). We further investigated the hypothesis that glutamate is the excitatory neurotransmitter at this synapse. The somata of sensory neurons in the pleural ganglia showed strong glutamate immunoreactivity. Very intense glutamate immunoreactivity was present in fibers within the neuropil and pleural-pedal connective. Localization of amino acids metabolically related to glutamate was also investigated. Moderate aspartate and glutamine immunoreactivity was present in somata of sensory neurons, but only weak labeling for aspartate and glutamine was present in the neuropil or pleural-pedal connective. In cultured sensory neurons, glutamate immunoreactivity was strong in the somata and processes and was very intense in varicosities; consistent with localization of glutamate in sensory neurons in the intact pleural-pedal ganglion. Cultured sensory neurons showed only weak labeling for aspartate and glutamine. Little or no gamma-aminobutyric acid or glycine immunoreactivity was observed in the pleural-pedal ganglia or in cultured sensory neurons. To further test the hypothesis that the sensory neurons use glutamate as a transmitter, in situ hybridization was performed by using a partial cDNA clone of a putative Aplysia high-affinity glutamate transporter. The sensory neurons, as well as a subset of glia, expressed this mRNA. Known glutamatergic motor neurons B3 and B6 of the buccal ganglion also appeared to express this mRNA. These results, in addition to previous physiological studies (Dale and Kandel [1993] Proc Natl Acad Sci USA. 90:7163-7167; Trudeau and Castellucci [1993] J Neurophysiol. 70:1221-1230; Armitage and Siegelbaum [1998] J Neurosci. 18:8770-8779)) establish glutamate as an excitatory neurotransmitter of the sensorimotor synapse.

Long-term changes in synthesis of intermediate filament protein, actin and other proteins in pleural sensory neurons of Aplysia produced by an in vitro analogue of sensitization training.

Electrical stimulation of peripheral nerves of isolated pleural-pedal ganglia, an in vitro analogue of long-term behavioral training in Aplysia, produced changes in the synthesis of specific proteins in pleural sensory neurons. The changes in incorporation of [35S]methionine into proteins occurring 24 h after electrical stimulation (late) were determined and compared with changes occurring immediately after stimulation (early). Eight proteins were affected 24 h after electrical stimulation. Three of these proteins were also affected immediately after electrical stimulation. Two of the proteins affected late are components of the cytoskeleton. One protein was identified as actin. The other protein was purified from preparative 2D-gels and partial amino acid sequences of 3 peptides derived from this protein were determined. The peptide sequences were found to be identical to those of an Aplysia intermediate filament protein.

Neural and molecular bases of nonassociative and associative learning in Aplysia.

A model that summarizes some of the neural and molecular mechanisms contributing to short- and long-term sensitization is shown in Figure 14. Sensitizing stimuli lead to the release of a modulatory transmitter such as 5-HT. Both serotonin and sensitizing stimuli lead to an increase in the synthesis of cAMP and the modulation of a number of K+ currents through protein phosphorylation. Closure of these K+ channels leads to membrane depolarization and the enhancement of excitability. An additional consequence of the modulation of the K+ currents is a reduction of current during the repolarization of the action potential, which leads to an increase in its duration. As a result, Ca2+ flows into the cell for a correspondingly longer period of time, and additional transmitter is released from the cell. Modulation of the pool of transmitter available for release (mobilization) also appears to occur as a result of sensitizing stimuli. Recent evidence indicates that the mobilization process can be activated by both cAMP-dependent protein kinase and protein kinase C. Thus, release of transmitter is enhanced not only because of the greater influx of Ca2+ but also because more transmitter is made available for release by mobilization. The enhanced release of transmitter leads to enhanced activation of motor neurons and an enhanced behavioral response. Just as the regulation of membrane currents is used as a read out of the memory for short-term sensitization, it also is used as a read out of the memory for long-term sensitization. But long-term sensitization differs from short-term sensitization in that morphological changes are associated with it, and long-term sensitization requires new protein synthesis. The mechanisms that induce and maintain the long-term changes are not yet fully understood (see the dashed lines in Fig. 14) although they are likely to be due to direct interactions with the translation apparatus and perhaps also to events occurring in the cell nucleus. Nevertheless, it appears that the same intracellular messenger, cAMP, that contributes to the expression of the short-term changes, also triggers cellular processes that lead to the long-term changes. One possible mechanism for the action of cAMP is through its regulation of the synthesis of membrane modulatory proteins or key effector proteins (for example, membrane channels). It is also possible that long-term changes in membrane currents could be due in part to enhanced activity of the cAMP-dependent protein kinase so that there is a persistent phosphorylation of target proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of neurons contributing to presynaptic inhibition in Aplysia californica.

Electrical stimulation of the connectives presynaptically inhibits the PSP from cell L10 to the left upper quadrant cells (LUQC). The present report describes the properties of some of the individual neurons contributing to this response. Action potentials produced in a cluster of cells in the abdominal ganglion reduce the amplitude of the L10-LUQC PSP for periods greater than 30 sec. At least some of their inhibitory action is mediated by a slow hyperpolarization of L10 which results in a decreased transmitter release. In other cases, however, the inhibition is produced with no significant alteration of L10 membrane potential, indicating that additional mechanisms may also be present. The neurons producing these effects are approximately 75 microns in diameter and are located on the left ventral surface of the ganglion. They have axons in the connectives and are thus activated by stimuli previously utilized to produce presynaptic inhibition. They appear to be some of the same cells that produce a slow inhibition of ink motoneuron L14; one of these has been identified as L32. The identification of these cells allows for the further biochemical, biophysical and morphological analysis of the events underlying presynaptic inhibition.

Simulation of the neural activity underlying a short-term modification of inking behavior in aplysia.

Carew and Kandel (1977) found that weak stimuli to the head or siphon fail to elicit the release of ink. When paired with each other, however, the second of the two leads to the release of ink. The present paper quantifies and simulates the neural events which underlie this short-term modification of the behavior. Noxious stimuli to the intact animal were mimicked by delivering trains of electrical stimuli to the connectives (conditioning input) and siphon nerve (test input) which drive the ink gland motor neurons located within the abdominal ganglion. Estimates of the synaptic conductance and equilibrium potential during the conditioning and test inputs were made and used to drive a previously developed Hodgkin-Huxley model of the ink motor neurons. The experimental and simulated results are in good agreement. Activation of one stimulus pathway augments or facilitates the ability of the other pathway to fire the ink motor neurons. The behavioral modification is causally related to a sustained synaptic current activated by the conditioning stimulus.

Slow depolarization produced by associative conditioning of Aplysia sensory neurons may enhance Ca2+ entry.

Sensory neurons activated by intracellular stimulation immediately before sensitizing tail shock displayed a slow depolarization after the shock. By contrast, sensory neurons exposed to the effects of tail shock alone or unpaired activation and tail shock showed a slow hyperpolarizing response to the shock. A voltage-sensitive Ca2+ conductance that is activated near the resting potential may be modulated by these opposite effects of associative and non-associative training.

Post-tetanic potentiation in Aplysia sensory neurons.

Post-tetanic potentiation (PTP) lasting several minutes is produced by high frequency intracellular activation of mechanosensory neurons. Control procedures suggest that the PTP involves a homosynaptic mechanism, restricted to the active synapses. The capacity for PTP appears to be a general property of mechanosensory neurons in this animal since it is found in separate sensory populations innervating the tail, head, and siphon of Aplysia.

Role of protein phosphatases in the modulation of neuronal membrane currents.

Although much is known regarding the modulation of ion channels through protein phosphorylation, little is known about their dephosphorylation by protein phosphatases (PrPs). To address this issue we examined the effects of a phosphatase inhibitor, okadaic acid (OA), and protein phosphatases obtained from rabbit skeletal muscle on steady-state membrane current in voltage-clamped sensory neurons of Aplysia. In addition, we examined the ability of OA and PrPs to modulate serotonin (5-HT)- and cAMP-induced inward currents and an opposite outward current induced by the peptide FMRFamide. The results suggest that: (1) some basal level of phosphorylation regulates membrane channels even in the absence of physiological stimulation; (2) the 5-HT-, cAMP-, and FMRFamide-induced responses are highly regulated by PrPs; and (3) the FMRFamide-induced outward current may be due at least in part to the activation of PrPs.