Conditioning and sensitization in the edible snail: the neurophysiological and metabolic characteristics]

Electrophysiological parameters and bound calcium (Ca(b)) level dynamics during sensitization development or conditioning of food aversion were studied in the command neurons of defense behaviour in the snail Helix lucorum. Responses evoked by a testing sensory stimulus were facilitated 50-60 min after the first sensitizing stimulation, while conditioned responses appeared 80-90 min after the first conditioning. It was the most essential electrophysiological difference between the long-term sensitization and conditioning. Analysis of the Ca(b)) dynamics in the neurons showed significant differences in calcium-dependent metabolism during the sensitization and conditioning, likely underlying the electrophysiological differences.     

The behavioral plasticity of the edible snail and its neuronal mechanisms]

Published results concerning behavioural habituation, sensitization, long-term processes, environmental conditioning, and conditioned food aversion in terrestrial snail Helix are discussed. Neural mechanisms underlying behavioural changes are considered. Problems of participation of motivational processed and development of plasticity in ontogenesis are reviewed.     

Learning-related long-term synaptic facilitation in snails: Possible mechanisms of long-term memory formation

The long-term changes in the electrical activity of command neurons related to sensitization and elaboration of associative defensive behavioral habit (food rejection) were studied inHelix snail. The long-term effects consisted of facilitation of synaptic components in neuronal responses to the test stimulations. Variations were found in the dynamics of long-term synaptic facilitation of responses to the applied chemical and tactile stimuli in the course of sensitization, as well as dependence of the degree of long-term facilitation of responses to the test stimulation at the site of its application with respect to the site of the sensitizing stimulation (site-specific sensitization). After conditioning, the synaptic response of command neurons to the conditioning stimulation appeared approximately 30 min later than did the long-term sensitization in these cells. The minimum duration of long-term synaptic facilitation of responses to the test stimulation varied from 1 h (for tactile stimulation) to 3 h (for chemical stimulation). The maximum duration of effects exceeded 4 h. It is suggested that the observed features of the synaptic plasticity in command neurons during learning are based on the selective regulation of synaptic inputs by specific protein regulators, whose lifespan does not exceed 1 h to 3 h.Neirofiziologiya/Neurophysiology, Vol. 25, No. 5, pp. 383–389, September–October, 1993.     

Conditioning as sensitization.

The concept of sensitization has a long history in psychology, and on both empirical and logical grounds it has been argued that sensitization and conditioning denote different neurophysiological processes. Investigators have devised elaborate control procedures that purport to differentiate sensitized and conditioned responses. But, mainly on logical grounds, it is argued that there is no valid basis for considering sensitization and conditioning as separate processes. A theory of learning is proposed in which sensitization is the basic construct.     

Specificity of molecular changes in neurons involved in memory storage

Evidence implicating molecular steps in memory storage is discussed, particularly with reference to molecular specificity and uniqueness and the possible relevance of these steps to other types of long-lasting transformations such as those of development, regeneration, and tumorigenesis. The role of protein kinase C-mediated phosphorylation of identified protein subtrates, such as a 20,000-dalton GTP-binding protein, is described for associative memory of the snail Hermissenda, associative conditioning of the rabbit, and long-term potentiation. Cyclic AMP-mediated phosphorylation during sensitization of the snail Aplysia is also examined.     

The architecture of the reflex arc]

The conceptual reflex arc in Helix is composed of identifiable local detectors, command neurons and modulatory neurons. The identification of neurons opens a perspective for identification of single synapses. The plastic changes within the reflex arc can be characterized as non-associative and associative. The non-associative plasticity (habituation and sensitization) is based on the dephosphorylation and phosphorylation of receptor and channel proteins. The associative plasticity (conditioning and extinction) is based on two levels of molecular modifications: the short-term changes underlined by the phosphorylation and dephosphorylation of receptor and channel molecules and the long-term changes referred to the expression of genes encoding structural and translocating proteins. The very selective receptor and channel modifications are due to the Hebbian plastic synapse supplemented with the principle of nonspecific (modulating) influences.     

Neurophysiological changes and dynamics of bound calcium during the development of associative learning in Helix lucorum

Neurophysiological effects and the dynamics of the content of bound calcium (Ca-b) in command neurons (LP1 and RP1) of defensive behavior during food aversion conditioning are studied inHelix lucorum. In the case of associative learning, there arises in these cells both a response to the conditioned stimulus and a nonspecific facilitation of the reactions to sensory stimuli that is characteristic for the state of sensitization. A response to the presentation of a conditioned stimulus is detected approximately 30 min after the development of long-term sensitization. The use of three or more paired stimuli reveals the characteristic dynamics of the level of Ca-b, which correlates with the neurophysiological effects and differs from the changes in Ca-b content during the development of "pure" sensitization. It is thought that the command neuron of defense behavior exhibits inHelix differences of the molecular-cellular mechanisms lying at the basis of the development of associative learning and sensitization.P. K. Anokhin Institute of Normal Physiology, Russian Academy of Sciences, Moscow. I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Neirofiziologiya, Vol. 24, No. 6, pp. 691–701, November–December, 1992.     

Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia

To study the effects of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.     

Inhibitor of protein synthesis blocks longterm behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia

To study the effect of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.     

Effect of 6-hydroxydopamine on the electrical characteristics of snail neurons in long-term sensitization

Studies of the influence of neurotoxin 6-hydroxydopamine selectively destroying the catecholamine terminals on long-term sensitization, and the role of dopamine in manifestation of characteristics of a membrane of identified neurons during elaboration of plasticity, were fulfilled. Injection of saline was used as the control. It is shown that preliminary injection of 6-hydroxydopamine reduces duration of long-term sensitization, but does not block it completely. It was shown that injection of 6-hydroxydopamine prevents diminishing of membrane and threshold potentials in withdrawal interneurons during formation of long-term sensitization. The experiments demonstrate that shift of electrical characteristics of withdrawal interneurons caused by injection of neurotoxin 6-hydroxydopamine to both naive snails and sensitized snails, statys during at least 10 days.     

Water-soluble and membrane proteins of the central nervous system of the edible snail at different stages of long-term sensitization

An increase in the amount of water-soluble proteins with the mobility of 0.56 in polyacrylamide gel was observed in the snail central nervous system at the early stages of long-term sensitization of the defensive reflex. Such an increase seems to be associated with the enhanced motor activity and a rise in the excitation level at this stage. 2 hours, 1 day and 3 days after long-term sensitization a dramatic decrease in the amount of water-soluble proteins with the mobility of 0.54, 0.42, 0.40 and membrane protein with the molecular weight of 24000 kD was observed. Such a decrease appears to reflect the stage nature of long-term sensitization and to be linked with shifts in the functional characteristics of some cellular elements of snail central nervous system.     

Selection on a haploid genotype for discrimination learning performance: Correlation between drone honey bees (Apis mellifera) and their worker progeny (Hymenoptera: Apidae)

Successful bidirectional selection for discriminative olfactory learning is reported for drone honey bees (Apis mellifera). Learning performance was evaluated using a discrimination conditioning procedure that required drones to discriminate between an appetitively reinforced odorant and one that was followed by punishment. Selective breeding produced high- and low-learning-performance lines of worker progeny that diverged from performance of workers whose fathers were selected at random. Furthermore, we show that levels of sucrose-induced sensitization are not correlated to learning performance. These results corroborate earlier findings and further demonstrate the power of selection on a haploid (drone) genotype. In addition, this study now shows that the demonstrated differences in learning performance cannot be completely accounted for by alteration of sucrose-induced sensitization. Thus, using this technique, it may be possible to select for associative conditioning without a pleiotropic increase in sensitization. The honey bee will be ideally suited to these types of correlation analyses in future studies.     

Vital investigation of the genome activity and neuronal synaptic plasticity in snail in the course of learning

Vital investigation of genome activity and its role in mechanisms of long-term synaptic plasticity formation were studied in LP11 neuron (command neurone of defence behaviour) during sensitisation (simple form of learning) in semi-intact preparation of snail Helix lucorum. Genome activity was investigate by means of specific fluorescent dye SYTO 16 and image analysis system. It was found that application of sensitizing stimulation (10% quinine solution) onto the snail head initiate two-phase changes in neural responses evoked by tactile or chemical stimulation. Depression of neural responses was obtained during short-term phase (during 1 hour after sensitization) and facilitation of neural responses--during long-term phases (over 1 hour). At the same time (15-20 min after learning), a significant increase in SYTO 16 fluorescent level was found in nucleus site LP11 neurone. Initial SYTO 16 fluorescence level was registered in 4-5 hours after sensitization. If the sensitization was produced during actinomycin D application (inhibitor of RNA synthesis, 20 microM) then facilitation of neural responses evoked by sensory stimulation were suppressed during long-term phase of sensitization and was the same as in control sensitized snails during short-term phase of learning. Increase in SYTO 16 fluorescence level in nucleus region site of LP11 neurone was completely prevented in sensitization during actinomycin D application. If actinomycin D was applied 30 min after sensitization (1 hour after first sensitizing stimulation) then synaptic facilitation and fluorescent dynamics was the same as in control sensitized snails. Our experimental data showed that nociceptive sensitization development was followed by quick (15-20 min) DNA activation and long-term synaptic facilitation (1 hour after sensitization), while induction of the processes was suppressed by inhibitor of RNA synthesis during short time interval (during 1 hour of learning).     

A pitfall for the expectancy theory of human eyelid conditioning

Two simple eyeblink conditioning experiments with random intermittent reinforcement schedules were performed. In Experiment 1, subjects had to rate their expectancy for an unconditioned stimulus (US) on a seven-level scale prior to each trial. As anticipated, expectancy for US increased with a successive conditioned stimulus (CS) alone, and decreased with successive CS-US pairings. However, Experiments 1 and 2 showed that the frequency of eyeblink conditioned responses (CRs) evolved in a direction opposite to that of expectancy changes: CRs increased, whereas expectancy for US decreased, and vice versa. The possible effect of sensitization on eyeblink response was ruled out by the lack of a run effect in an unpaired control group in Experiment 2. These results tend to disconfirm the expectancy theory of conditioning. Although they were explicitly predicted by the conventional "strength" theory of conditioning, an alternative interpretation is proposed within a cognitive framework.     

Behavioral changes during the acquisition of long-term sensitization of the defensive reflex in the edible snail

Reaction of long-term sensitization (LTS) of defensive reflex was elaborated in the snail. It was accompanied by a considerable increase of the time of closing of pneumostome. The results show that LTS is preserved for more than two week and may be a model of long-term memory.     

Cell and molecular analysis of long-term sensitization in Aplysia

We have found that one cellular locus for the storage of the memory underlying short-term sensitization of the gill and siphon withdrawal reflex in Aplysia is the set of monosynaptic connections between the siphon sensory cells and the gill and siphon motor neurons. These connections also participate in the storage of memory underlying long-term sensitization. In animals that have undergone long-term sensitization, the amplitudes of the monosynaptic connections are significantly larger (2.2x) than the ones in control animals. To study the mechanisms of onset and retention of long-term synaptic facilitation that underly long-term sensitization and the role of protein synthesis in long-term memory, we have developed two types of reduced preparations: the intact reflex isolated from the remainder of the animal, and a dissociated cell culture system in which the monosynaptic component (sensory neurons and motor neurons) of the neuronal circuit mediating the withdrawal reflex is reconstituted. We found that protein synthesis inhibitors, such as anisomycin or emetine, and RNA synthesis inhibitors, such as actinomycin D or alpha-amanitin, blocked long-term facilitation without interfering with short-term facilitation. These results suggest that the acquisition of long-term memory may require the expression of genes and the synthesis of proteins not needed for short-term memory.     

Neuronal mechanisms contributing to long-term sensitization in Aplysia

1.) Cellular processes that contribute to the acquisition and expression of long-term sensitization have been examined in Aplysia. The tail-siphon withdrawal reflex was studied because the neural circuit for this reflex has been well characterized. Furthermore, the sensory neurons of this neural circuit exhibit cellular changes that accompany short-term sensitization. 2.) Repeated application of noxious stimuli to the animal produces a long-lasting enhancement of reflex withdrawal of the siphon when the animal is tested with a weak stimulus to the tail. These findings confirm the existence of long-term sensitization in Aplysia, first described by Pinkser et al. (1973). 3.) Biophysical correlates of long-term sensitization were examined in the first central relay of the tail-siphon reflex circuit, the sensory neurons that innervate the animal's tail. The net outward membrane currents of these cells reduced after 24 hours as a consequence of long-term sensitization training. 4.) The intracellular signal for the induction of these changes in membrane currents was examined by intracellular injection of cAMP into individual sensory neurons. This procedure mimics at least some of the effects of sensitization training at the single-cell level. cAMP induced a long-term reduction of membrane K+ currents 24 hours after the cells were injected with cAMP. The membrane currents reduced by cAMP were similar to those reduced by long-term sensitization training. 5.) Preliminary experiments indicate that neurotransmitters and agents that induce an evaluation of cAMP in the sensory neurons also alter the incorporation of labeled amino acids into specific proteins in the sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular mechanisms underlying emotional learning and memory in the lateral amygdala

Fear conditioning is a valuable behavioral paradigm for studying the neural basis of emotional learning and memory. The lateral nucleus of the amygdala (LA) is a crucial site of neural changes that occur during fear conditioning. Pharmacological manipulations of the LA, strategically timed with respect to training and testing, have shed light on the molecular events that mediate the acquisition of fear associations and the formation and maintenance of long-term memories of those associations. Similar mechanisms have been found to underlie long-term potentiation (LTP) in LA, an artificial means of inducing synaptic plasticity and a physiological model of learning and memory. Thus, LTP-like changes in synaptic plasticity may underlie fear conditioning. Given that the neural circuit underlying fear conditioning has been implicated in emotional disorders in humans, the molecular mechanisms of fear conditioning are potential targets for psychotherapeutic drug development.     

DOES DIZOCILPINE (MK-801) INHIBIT THE DEVELOPMENT OF MORPHINE-INDUCED BEHAVIOURAL SENSITIZATION IN RATS?

Intermittent morphine pretreatment (10 mg/kg/day for 14 days) induced long-lasting (one month post-treatment) sensitization to the locomotor effects of morphine and amphetamine in rats. Co-administration of the non-competitive NMDA-receptor antagonist dizocilpine (MK-801) (0.1 mg/kg) with morphine did not prevent the development of long-term behavioural sensitization. However, this dose of MK-801 did cause long-term sensitization to its own locomotor effects. Co-administration of 0.25 mg/kg MK-801 with morphine caused death in 60% of the animals. In the animals that survived MK-801 plus morphine pretreatment, neither short-term (3 days) nor long-term morphine-induced sensitization was observed. MK-801 alone (0.25 mg/kg/day for 14 days) induced short-term cross-sensitization to morphine. Thus, the development of long-term morphine-induced locomotor sensitization could only be prevented by a dose of MK-801 that yields a lethal combination with morphine. In addition, MK-801 induced sensitization to its own locomotor effects and cross-sensitization to morphine. These findings seriously question whether MK-801 can be used to study the development of morphine-induced behavioural sensitization. © 1997 Elsevier Science Inc.     

A functional analysis of social reinforcement in vicarious verbal conditioning

This article reports the results of 629 subjects in three experiments designed to replicate and extend the phenomenon of vicarious verbal conditioning. Experiment I replicated the finding that subjects who responded most to vicarious verbal conditioning were aware of the contingency involved. Experiment II attempted to examine the effects of prior history with the verbal reinforcer on vicarious verbal conditioning by providing seven groups of subjects with varying classic conditioning histories prior to vicarious verbal conditioning. The null results associated with this experiment were hypothesized to be due to the fact that the vicarious verbal conditioning took place in a language laboratory where the subjects could hear but not see the model. Experiment III replicated Experiment II in a live group context as was done in Experiment I. The results showed that vicarious verbal conditioning was again found to take place, that associating the verbal reinforcer with a tone or tone plus money via forward classic conditioning potentiated the effects of the verbal reinforcer, that backward classic conditioning did not potentiate the reinforcer, d) nor did either of two sensitization procedures potentiate the effects of the verbal reinforcer. Both aware and unaware subjects evidenced vicarious verbal conditioning.