Number Of Conditioning Trials Research Articles

Species- and size-related differences in dopamine-like immunoreactive clusters in the brain of Nasonia vitripennis and N. giraulti

An extreme reduction in body size has been shown to negatively impact the memory retention level of the parasitic wasp Nasonia vitripennis. In addition, N. vitripennis and Nasonia giraulti, closely related parasitic wasps, differ markedly in the number of conditioning trials required to form long-term memory. These differences in memory dynamics may be associated with differences in the dopaminergic neurons in the Nasonia brains. Here, we used dopamine immunoreactivity to identify and count the number of cell bodies in dopaminergic clusters of normal- and small-sized N. vitripennis and normal-sized N. giraulti. We counted in total a maximum of approximately 160 dopaminergic neurons per brain. These neurons were present in 9 identifiable clusters (D1a, D1b, D2, D3, D4a, D4b, D5, D6 and D7). Our analysis revealed that N. giraulti had fewer cells in the D2 and D4a clusters but more in D4b, compared with normal-sized N. vitripennis. In addition, we found fewer cells in the D5 and D7 cluster of small-sized N. vitripennis compared to normal-sized N. vitripennis. A comparison of our findings with the literature on dopaminergic clusters in the fruit fly Drosophila melanogaster and the honey bee Apis mellifera indicates that clusters D2, D3 and D5 may play a role in memory formation in Nasonia wasps. The results from both the species comparison and the size comparison are therefore of high interest and importance for our understanding of the complex intricacies that underlie the memory dynamics of insects.

Differentially expressed genes linked to natural variation in long-term memory formation in Cotesia parasitic wasps.

Even though learning and memory are universal traits in the Animal Kingdom, closely related species reveal substantial variation in learning rate and memory dynamics. To determine the genetic background of this natural variation, we studied two congeneric parasitic wasp species, Cotesia glomerata and C. rubecula, which lay their eggs in caterpillars of the large and small cabbage white butterfly. A successful egg laying event serves as an unconditioned stimulus (US) in a classical conditioning paradigm, where plant odors become associated with the encounter of a suitable host caterpillar. Depending on the host species, the number of conditioning trials and the parasitic wasp species, three different types of transcription-dependent long-term memory (LTM) and one type of transcription-independent, anesthesia-resistant memory (ARM) can be distinguished. To identify transcripts underlying these differences in memory formation, we isolated mRNA from parasitic wasp heads at three different time points between induction and consolidation of each of the four memory types, and for each sample three biological replicates, where after strand-specific paired-end 100 bp deep sequencing. Transcriptomes were assembled de novo and differential expression was determined for each memory type and time point after conditioning, compared to unconditioned wasps. Most differentially expressed (DE) genes and antisense transcripts were only DE in one of the LTM types. Among the DE genes that were DE in two or more LTM types, were many protein kinases and phosphatases, small GTPases, receptors and ion channels. Some genes were DE in opposing directions between any of the LTM memory types and ARM, suggesting that ARM in Cotesia requires the transcription of genes inhibiting LTM or vice versa. We discuss our findings in the context of neuronal functioning, including RNA splicing and transport, epigenetic regulation, neurotransmitter/peptide synthesis and antisense transcription. In conclusion, these brain transcriptomes provide candidate genes that may be involved in the observed natural variation in LTM in closely related Cotesia parasitic wasp species.

Differences in long-term memory stability and AmCREB level between forward and backward conditioned honeybees (Apis mellifera).

In classical conditioning a predictive relationship between a neutral stimulus (conditioned stimulus; CS) and a meaningful stimulus (unconditioned stimulus; US) is learned when the CS precedes the US. In backward conditioning the sequence of the stimuli is reversed. In this situation animals might learn that the CS signals the end or the absence of the US. In honeybees 30 min and 24 h following backward conditioning a memory for the excitatory and inhibitory properties of the CS could be retrieved, but it remains unclear whether a late long-term memory is formed that can be retrieved 72 h following backward conditioning. Here we examine this question by studying late long-term memory formation in forward and backward conditioning of the proboscis extension response (PER). We report a difference in the stability of memory formed upon forward and backward conditioning with the same number of conditioning trials. We demonstrate a transcription-dependent memory 72 h after forward conditioning but do not observe a 72 h memory after backward conditioning. Moreover we find that protein degradation is differentially involved in memory formation following these two conditioning protocols. We report differences in the level of a transcription factor, the cAMP response element binding protein (CREB) known to induce transcription underlying long-term memory formation, following forward and backward conditioning. Our results suggest that these alterations in CREB levels might be regulated by the proteasome. We propose that the differences observed are due to the sequence of stimulus presentation between forward and backward conditioning and not to differences in the strength of the association of both stimuli.

Modulation of ethanol-induced conditioned place preference in mice by 3-amino-1,2,4-triazole and D-penicillamine depends on ethanol dose and number of conditioning trials

Previous studies have shown that both 3-amino-1,2,4-triazole (AT), which inhibits metabolism of ethanol (EtOH) to acetaldehyde by inhibiting catalase, and D-penicillamine (D-P), an acetaldehyde-sequestering agent, modulate EtOH-conditioned place preference (CPP) in male albino Swiss (IOPS Orl) mice. These studies followed a reference-dose-like procedure, which involves comparing cues that have both been paired with EtOH. However, the role of EtOH-derived acetaldehyde has not been examined using a standard CPP method, and efficacy of these treatments could be different under the two circumstances. In the present investigation, we manipulated the strength of CPP across five separate studies and evaluated the effect of D-P and AT on EtOH-induced CPP following a standard unbiased CPP procedure. Mice received pairings with vehicle-saline injections with one cue and, alternatively, with AT- and D-P-EtOH with another cue. Our studies indicate that AT and D-P only disrupt CPP induced by EtOH in mice when the number of conditioning sessions and the dose of EtOH are low. These findings suggest that acquisition of EtOH-induced CPP may depend on the levels of acetaldehyde available during memory acquisition and the strength of the memory. Therefore, we propose that, at least when the memory processes are labile, brain acetaldehyde could participate in the formation of Pavlovian learning elicited by EtOH.

A Computational Framework for Understanding Decision Making through Integration of Basic Learning Rules

Nonassociative and associative learning rules simultaneously modify neural circuits. However, it remains unclear how these forms of plasticity interact to produce conditioned responses. Here we integrate nonassociative and associative conditioning within a uniform model of olfactory learning in the honeybee. Honeybees show a fairly abrupt increase in response after a number of conditioning trials. The occurrence of this abrupt change takes many more trials after exposure to nonassociative trials than just using associative conditioning. We found that the interaction of unsupervised and supervised learning rules is critical for explaining latent inhibition phenomenon. Associative conditioning combined with the mutual inhibition between the output neurons produces an abrupt increase in performance despite smooth changes of the synaptic weights. The results show that an integrated set of learning rules implemented using fan-out connectivities together with neural inhibition can explain the broad range of experimental data on learning behaviors.

How the number of learning trials affects placebo and nocebo responses

Conditioning procedures are used in many placebo studies because evidence suggests that conditioning-related placebo responses are usually more robust than those induced by verbal suggestions alone. However, it has not been shown whether there is a causal relation between the number of conditioning trials and the resistance to extinction of placebo and nocebo responses. Here we test the effects of either one or four sessions of conditioning on the modulation of both non-painful and painful stimuli delivered to the dorsum of the foot. Placebo and nocebo manipulations were obtained by pairing green or red light to a series of stimuli that were made lower or higher with respect to a yellow light associated with a series of control stimuli. Subjects were told that the lights would indicate a treatment that would reduce or increase non-painful and painful stimuli to the foot. They were randomly assigned to either Group 1 or 2. Group 1 underwent one session of conditioning and Group 2 received four sessions of conditioning. We found that one session of conditioning (Group 1) induced nocebo responses, but not placebo responses in no pain condition. After one session of conditioning, we observed both nocebo and placebo responses to painful stimulation. However, these effects extinguished over time. Conversely, four sessions of conditioning (Group 2) induced robust placebo and nocebo responses to both non-painful and painful stimuli that persisted over the entire experiment. These findings suggest that the strength of learning may be clinically important for producing long-lasting placebo effects.

Cannabinoid conditioned reward and aversion: behavioral and neural processes.

The discovery that delta-9-tetrahydrocannabinol (Δ(9)-THC) is the primary psychoactive ingredient in marijuana prompted research that helped elucidate the endogenous cannabinoid system of the brain. Δ(9)-THC and other cannabinoid ligands with agonist action (CP 55,940, HU210, and WIN 55,212-2) increase firing of dopamine neurons and increase synaptic dopamine in brain regions associated with reward and drug addiction. Such changes in cellular processes have prompted investigators to examine the conditioned rewarding effects of the cannabinoid ligands using the place conditioning task with rats and mice. As reviewed here, these cannabinoid ligands can condition place preferences (evidence for rewarding effects) and place aversions (evidence for aversive qualities). Notably, the procedural details used in these place conditioning studies have varied across laboratories. Such variation includes differences in apparatus type, existence of procedural biases, dose, number of conditioning trials, injection-to-placement intervals, and pre-training drug exposure. Some differences in outcome across studies can be explained by these procedural variables. For example, low doses of Δ(9)-THC appear to have conditioned rewarding effects, whereas higher doses have aversive effects that either mask these rewarding effects or condition a place aversion. Throughout this review we highlight key areas that need further research.

Within-subject effects of number of trials in rat conditioning procedures.

D. A. Gottlieb (2008) reported finding no effects of number of conditioning trials in a series of between-subjects magazine approach experiments, when number of sessions and total training time were held constant. This article reports 7 comparable within-subject experiments that looked for effects of number of trials in a variety of conditioning preparations. Experiments 1-3 detected effects of multiplying the number of trials by factors of 4 and 8 in a conditioned magazine approach procedure in which visual and auditory stimuli were paired with food. Experiments 4A and 4B detected effects of a factor of 4 trials in a conditioned flavor preference procedure in which flavors were paired with polycose. Experiments 5 and 6 detected an effect of a factor of 3 trials in a conditioned taste aversion procedure and in a fear conditioning procedure, respectively. Results suggest that variables other than number of trials might play important roles in determining the acquisition of conditioned responding, but effects of number of trials can be detected with sensitive enough procedures.

Olfactory conditioning of the sting extension reflex in honeybees: Memory dependence on trial number, interstimulus interval, intertrial interval, and protein synthesis

Harnessed bees learn to associate an odorant with an electric shock so that afterward the odorant alone elicits the sting extension response (SER). We studied the dependency of retention on interstimulus interval (ISI), intertrial interval (ITI), and number of conditioning trials in the framework of olfactory SER conditioning. Forward ISIs (conditioned stimulus [CS] before unconditioned stimulus [US]) supported higher retention than a backward one (US before CS) with an optimum around 3 sec. Spaced trials (ITI 10 min) supported higher retention than massed trials (ITI 1 min) and led to the formation of a late long-term memory (l-LTM) that depended on protein synthesis. Our results reaffirm olfactory SER conditioning as a reliable tool for the study of learning and memory.

Not What You Expect: Experience but not Expectancy Predicts Conditioned Responses in Human Visual and Supplementary Cortex

When paired with aversive events, visual conditioned stimuli (CS) provoke increased activations in visual cortex. It is unclear however whether these changes reflect cognitive processes such as expectancy of the aversive unconditioned stimulus (US), or implicit associative learning of the contingencies outside awareness. Here, we used the "gambler's fallacy" phenomenon to parametrically and inversely manipulate the expectancy of an US and the number of conditioning trials: Increasing the number of CS-US pairings was associated with participants expecting the US to be less likely and vice versa. Magnetocortical activity evoked by the CS in occipital and supplementary motor areas was linearly related to the associative strength (number of CS-US pairings), but decreased as a function of expectancy. These results suggest that the cortical facilitation of fear cue processing is determined by associative strength and previous exposure to learning contingencies rather than by the cognitive anticipation for the US.

Intravenous nicotine conditions a place preference in rats using an unbiased design

The rewarding effects of nicotine contribute to the chronic use of tobacco products. The place conditioning task, a widely used pre-clinical model to study drug reward, has lead to mixed results in rats when nicotine was administered subcutaneously or intraperitoneally; intravenously administered nicotine has not been examined. Further, much of the research demonstrating a nicotine-conditioned place preference in rats has used a biased design making these results susceptible to non-reward interpretations. The present study assessed whether intravenous (IV) nicotine would condition a place preference in an unbiased design and evaluated important behavioral parameters: nicotine dose, number of conditioning trials, and infusion-to-placement interval. In adult male Sprague Dawley rats, IV nicotine (0.03 mg/kg) conditioned a place preference after 8 conditioning trials. This conditioned preference was observed whether nicotine was infused 10 min before or immediately after placement in the paired environment for 10 min; infusing nicotine immediately after removal from the paired environment did not condition a preference after 4 or 8 conditioning trials. Four conditioning trials were not sufficient to condition a preference regardless of the temporal relation between the paired environment and 0.03 mg/kg nicotine. A 0.01 mg/kg dose of nicotine did not condition a place preference after 4 or 8 trials when infused immediately upon placement in the paired environment. Intravenous nicotine (0.03 mg/kg) has rewarding effects in an unbiased design suggesting that the place conditioning protocol used in the present study might be an especially useful model for studying the processes underlying the conditioned rewarding effects of nicotine.

Conditioned observation of books and accelerated acquisition of textual responding by preschool children.

We report an experiment investigating the effects of conditioning books as reinforcers for observing responses on the learning of textual responses by pre-school children. The independent variable was the acquisition of conditioned reinforcement of observing responses and choice of book stimuli in free play settings where children could choose to play with toys or look at books. Prior to the conditioning procedures, the children played with toys and did not look at books in free time in 4 preconditioning 5minute free-play sessions. During the treatment we conducted simultaneous stimulus conditioning procedures until looking at books became the preferred free-play activity. The dependent variable consisted of the numbers of learn-units-to mastery of textual responses before and after conditioning books as reinforcers for observing responses. Three boys and one girl (ages 2 years and 9 months to 4 years) participated in the experiment in a pre and post learn-units-to-criterion and simultaneous matched-pairs design with a time-lagged component. Prior to reinforcement conditioning, we matched children in pairs based on learn units they required to master a sets of 5 counterbalanced word sets. One child in each of 2 pairs received book conditioning initially, and another child received an equal number of conditioning trials with toys, as a control conditions, and then book conditioning. The results showed that (1) all four children required fewer learn-units-to-criterion on textual responses after books were conditioned as reinforcers for choice and observing and those who received the book conditioning first performed best. (2) Three of the four children maintained preference for books at 33%, 83%, and 100% of time in free play probes at 1 month. (3) There were no maintenance effects on accuracy of textual responding.

Influence of the dose and the number of drug-context pairings on the magnitude and the long-lasting retention of cocaine-induced conditioned place preference in C57BL/6J mice

The place conditioning procedure is increasingly used to study relapse in drug seeking in mice. However, the retention course of drug-induced place preference has not been systematically characterized. The effects of cocaine doses and number of conditioning trials on both the magnitude and the persistence of cocaine-induced conditioned place preference (CPP) were investigated in C57BL/6J mice. Twelve groups of animals were injected with saline, 4, 8 or 12 mg/kg cocaine (i.p.) and submitted to an unbiased counterbalanced place conditioning protocol including one, two or four drug-pairing sessions. Subsequently, the animals were tested at various time intervals after the last conditioning session. One cocaine-pairing session was insufficient to induce a CPP. Two and four pairing sessions resulted in significant place preferences of similar magnitude for all tested doses of cocaine, the place preference induced by the greatest number of pairing sessions being the strongest. In the two-pairing groups, place preference lasted less than 14 days for any tested dose of cocaine. In contrast, all four-pairing groups still showed significant place preference 28 days after the last conditioning session. However, the magnitude of cocaine place preference slowly declined at a rate that was dependent upon cocaine dose. On the 35-day post-conditioning interval, only the 12-mg/kg cocaine group still displayed a significant place preference, whereas place preference was undetectable at 42 and 56 days post-conditioning for all groups. The number of cocaine-pairing sessions, but not cocaine dose, affected the magnitude of cocaine place preference in mice when tested 1 day after the last conditioning session. In contrast, both cocaine doses and the number of pairing sessions affected the persistence of cocaine place preference. Overall, these results demonstrate that cocaine-induced place preference is a long lasting phenomenon that is strongly affected by the number of drug-pairing trials.

Enhancement of antibody response by one-trial conditioning: Contrasting results using different antigens

New research in conditioned enhancement of antibody response requires a general paradigm effective with different antigens. In this experiment series we applied a one-trial protocol using keyhole limpet hemocyanin immunization as an unconditioned stimulus. Several different conditions were tested. Two different times between conditioning and test trial, two relevant antigen doses and the use of an antigen booster during test trial were investigated. We did not find a conditioned effect in any of the conditions used. In contrast, we found a reliable albeit modest conditioned effect using hen egg lysozyme as unconditioned stimulus. By comparing these and other findings we conclude that the number of conditioning trials is a possible requirement for a more reliable conditioning of antibody response.

Massed and spaced learning in honeybees: the role of CS, US, the intertrial interval, and the test interval.

Conditioning the proboscis extension reflex of harnessed honeybees (Apis mellifera) is used to study the effect temporal spacing between successive conditioning trials has on memory. Retention is monitored at two long-term intervals corresponding to early (1 and 2 d after conditioning) and late long-term memory (3 and 4 d). The acquisition level is varied by using different conditioned stimuli (odors, mechanical stimulation, and temperature increase at the antenna), varying strengths of the unconditioned stimulus (sucrose), and various numbers of conditioning trials. How learning trials are spaced is the dominant factor both for acquisition and retention, and although longer intertrial intervals lead to better acquisition and higher retention, the level of acquisition per se does not determine the spacing effect on retention. Rather, spaced conditioning leads to higher memory consolidation both during acquisition and later, between the early and long-term memory phases. These consolidation processes can be selectively inhibited by blocking protein synthesis during acquisition.

Prolonged activation of cAMP-dependent protein kinase during conditioning induces long-term memory in honeybees.

To investigate the function cAMP-dependent protein kinase (PKA) exerts in the induction of long-term memory, changes in PKA activity induced by associative learning in vivo were measured in the antennal lobes (ALs) of honeybees. The temporal dynamics of PKA activation depend on both the sequence of conditioned and unconditioned stimuli and the number of conditioning trials. Only multiple-trial conditioning, which induces long-term memory (LTM), leads to a profound prolongation of PKA activation mediated by the NO/cGMP system. Imitation of this prolonged PKA activation in the ALs in combination with single-trial conditioning is sufficient to induce LTM. These findings not only demonstrate the close connection between conditioning procedure and temporal dynamics in PKA activation but also reveal that already during conditioning a distinct temporal pattern of PKA activation is critical for LTM induction in intact animals.

Potentiation of phototactic suppression in Hermissenda by a chemosensory stimulus during compound conditioning.

Modifications of Hermissenda's phototactic behavior by compound pairings of light, scallop extract, and rotation were assessed. In general, the scallop extract potentiated phototactic suppression. Potentiation was dependent on (a) conjunctive presentations of scallop and light, (b) number of conditioning trials, and (c) scallop extract concentration. In related experiments, no second-order conditioning or sensory preconditioning of phototactic suppression was observed, indicating that within-compound associations did not contribute appreciably to potentiation. These results represent the first detailed analysis of compound conditioning in a mollusk using discrete presentations of well-characterized conditioned stimuli from distinct sensory modalities.

Relationship between number of conditioning trials per training session and avoidance learning in horses

Sixteen horses were used to determine if number of trials given per training session (5, 10, 15 or 20) affected learning performance in an avoidance conditioning task. The horse had to move from one side of a test pen to the other during an auditory cue presentation to avoid aversive stimulation. A pen 8 m×3.6 m, divided into two equal sections by a 13-cm diameter plastic pipe lying on the ground, was used as the test pen. Painted plywood panels were fastened to the fence in half the pen to help horses distinguish visually between the two parts. A 10-s auditory cue was used as a signal for horses to move from one side of the test pen to the other. A 20-s intertrial interval was used. Training sessions were conducted every third day. Each trial was recorded as an avoidance (the horse completed the task during auditory cue presentation and avoided aversive stimulus) or an error (the horse received aversive stimulus). After completing ten consecutive avoidances (criterion), the horse was removed from the study. Numbers of training sessions, trials, avoidances and errors until reaching criterion were recorded for each horse. Horses varied greatly within these variables with ranges of 3–18 sessions, 37–121 trials, 20–68 avoidances and 17–53 errors to criterion. No differences were detected ( P>0.05) in the number of conditioning trials per training session (treatment) for the mean number of trials, avoidances or errors to criterion. Number of training sessions to criterion differed ( P<0.01) among treatments, indicating that an optimum number of learning trials per training session might exist. Mean sessions to criterion for horses receiving 5, 10, 15 and 20 trials per session were 15.1±1.3, 5.8±1.1, 5.3±1.1 and 4.6±1.1, respectively. Regression analysis indicated that 16.2 trials per training session would minimize number of sessions to criterion. Although it is widely assumed that learning efficiency in horses is decreased when intense activity is concentrated into a small number of sessions, these results indicate that moderate repetition of training activities is needed for efficient learning.

Angiotensin II mediates a conditioned taste aversion in water-replete rats

Many compounds that exert an influence on behavioral processes will, under the unique conditions of the conditioned taste aversion (CTA) procedure, cause animals to avoid consuming an otherwise preferred fluid. While angiotensin II (ANG II) is a peptide with a number of behavioral and physiological actions, previous research did not support its role as an agent capable of inducing a CTA. Those studies used fluid deprivation to induce fluid intake and only a single conditioning trial. Fluid deprivation can elevate endogenous ANG II levels that may have interfered with or masked the ability of ANG II injections to exert an effect as a CTA-inducing agent. The present study reassessed the ability of ANG II to induce a CTA using fully hydrated animals and a number of conditioning trials. ANG II was able to induce a significant taste aversion at a dose five times lower than that used in previous studies.

Effect of number of conditioning trials on the development of associative tolerance to morphine.

The acquisition of associative tolerance to the analgesic effects of morphine was investigated by giving independent groups of rats 1, 3, 5, 8, 14, 20, or 30 administrations of drug either explicitly paired or unpaired with a distinctive context. Tolerance, assessed on a tail-flick device using dose-response curve (DRC) methodology, developed more rapidly and reached greater magnitude when morphine and the distinctive context were explicitly paired rather than explicitly unpaired. Tolerance magnitude in both conditions reached a maximum at eight conditioning sessions. It is argued that the tolerance found in both treatment groups was associatively controlled. The function of handling and injection cues as conditioned stimuli, and the deleterious effects of latent inhibition and partial reinforcement on conditioned excitation and conditioned inhibition are discussed.