桔小实蝇雄成虫联系性嗅觉学习

【目的】为了探究桔小实蝇 Bactrocera dorsalis (Hendel)雄成虫的嗅觉学习能力。【方法】本研究采用经典性嗅觉条件反射训练法（classical olfactory conditioning）在室内对固定的羽化后14-17日龄的桔小实蝇雄成虫进行气味与食物的联合学习训练, 即薄荷精油和10%蔗糖溶液联合的奖赏性训练（appetitive conditioning）以及甲基丁香酚(methyl eugenol, ME)和饱和盐溶液联合的惩罚性训练（aversive conditioning）,并以伸喙反射行为（proboscis extension reflex, PER）作为学习与否的判定标准。【结果】经过奖赏性训练后,桔小实蝇雄成虫对薄荷精油的伸喙反射率可从0%增加至68%;而经过惩罚性训练后,桔小实蝇对甲基丁香酚的伸喙反射率可从100%降低至36.54%,且这种伸喙反射率的变化是通过气味条件刺激（conditioned stimulus）和食物非条件刺激（unconditioned stimulus）的对称性联合而产生的。【结论】结果表明,桔小实蝇雄性成虫具有较强的联系性嗅觉学习能力,并且两种刺激的联合是形成学习记忆的必要条件。     

Hippocampal place cells acquire location-specific responses to the conditioned stimulus during auditory fear conditioning

We recorded neurons from the hippocampus of freely behaving rats during an auditory fear conditioning task. Rats received either paired or unpaired presentations of an auditory conditioned stimulus (CS) and an electric shock unconditioned stimulus (US). Hippocampal neurons (place and theta cells) acquired responses to the auditory CS in the paired but not in the unpaired group. After CS-US pairing, rhythmic firing of theta cells became synchronized to the onset of the CS. Conditioned responses of place cells were gated by their location-specific firing, so that after CS-US pairing, place cells responded to the CS only when the rat was within the cell's place field. These findings may help to elucidate how the hippocampus contributes to context-specific memory formation during associative learning.     

Conditioned augmentation of natural killer cell activity. Independence from nociceptive effects and dependence on interferon-beta

Injection of mice with 20 micrograms polyinosinic: polycytidylic acid (Poly I:C) after exposure to camphor odor results in a conditioned augmentation of natural killer cell (NK) activity. In this study, we show that the conditioned response is not the result of nociceptive stimulation and that interferon-beta (IFN), but not IFN-alpha can replace Poly I:C as the unconditioned stimulus (US). Two conditioned stimuli (CS) were used with equivalent results. A combination CS consisting of a novel taste (saccharin) and a 125 mg/kg injection of LiCl that induces gastric upset was paired with a Poly I:C or IFN-beta (1 X 10(4) IU) injection. This resulted in an augmentation of NK activity when the conditioned animals were reexposed to the saccharin-LiCl CS. In addition, an identical conditioned response was elicited when a camphor odor CS was paired with either of these US. To test whether the conditioned response might be an artifact not detected by our controls, a mock conditioning experiment was performed, which assessed the differential effect of multiple exposures to the saccharin-LiCl CS without a CS/US pairing. The mock conditioned group was significantly suppressed relative to saline treated controls, whereas the mock nonconditioned group and the mock conditioned group that was not reexposed to the CS after conditioning did not show significant suppression. This indicates that the augmentation observed in the conditioned group after CS/US pairing was not the result of exposure to the CS itself. Small doses of Poly I:C (5 micrograms or 2.5 micrograms) given on days 3 and 5 (or on day 5 only) to boost NK activity had the effect of increasing the magnitude of the conditioned response measured on day 6. In addition, an identical conditioned response was observed when the interval between the CS/US pairing and the later CS exposures was changed, which places the test for the conditioned response either on day 6 (CS given on days 3 and 5) or day 10 (CS given on days 7 and 9). These results show that the observed conditioned enhancement of NK activity in conditioned animals is not caused by any nociceptive properties of the CS itself and is dependent on the IFN-beta produced after Poly I:C injection in the conditioned paradigm.     

Conditioning odor-shock associations in the black blowfly,Phormia regina

Discriminative classical conditioning of an olfactory avoidance response was demonstrated in the blowfly, Phormia regina.Learning indices were calculated as the fraction of flies avoiding the CS+ (conditioned stimulus paired with electric shock reinforcement) minus the fraction of flies avoiding the CS-(conditioned stimulus not paired with electric shock), averaged over two different groups of flies, in which reciprocal odors were used as the CS+. Avoidance responses to both odors presented simultaneously at a T-maze choice point yielded mean learning indices of zero for naive flies or pseudoconditioned (shock alone) or sensitized (odors alone) controls. In contrast, pairing an odor with electric shock produced a mean learning index significantly greater than zero. These results are similar to those reported for Drosophila melanogaster.Blow flies, however, showed much lower levels of associative learning than fruit flies.     

Neural-network simulations of two context-dependence phenomena

This paper describes simulations of two context-dependence phenomena in Pavlovian conditioning, using a neural-network model that draws on knowledge from neuroscience and makes no distinction between operant and respondent learning mechanisms. One phenomenon is context specificity or the context-shift effect, the decrease of conditioned responding (CR) when the conditioned stimulus (CS) is tested in a context different from the one in which it had been paired with the unconditioned stimulus (US). The other effect is renewal, the recovery of CR in the training context after extinction in another context. For specificity (simulation 1), two neural networks were first given 200 CS-US pairings in a context. Then, the CS was tested either in the training context or a new context. Output activations in the new context were substantially lower. For renewal (simulation 2), two networks were first given 200 CS-US pairings in a context, then 100 extinction trials in either the same context or a new one, and then tested back in the training context. Output activations during the test phase were substantially higher after extinction in a new context. The results are interpreted in terms of the dynamics of activations and weights.     

Classical conditioning of breathing pattern after two acquisition trials in 2-day-old mice.

The aim of the present study was to test whether breathing pattern conditioning may occur just after birth. We hypothesized that sensory stimuli signaling the resumption of maternal care after separation may trigger an arousal and/or orienting response accompanied with concomitant respiratory changes. We performed a conditioning experiment in 2-day-old mice by using an odor (lemon) as the conditioned stimulus (CS) and maternal care after 1 h without the mother as the unconditioned stimulus (US). Each pup underwent two acquisition trials, in which the CS was presented immediately before (experimental paired group, n = 30) or 30 min before (control unpaired group, n = 30) contact with the mother. Conditioning was tested by using noninvasive whole body plethysmography to measure the respiratory response to the CS for 1 min. We found significantly stronger respiratory responses to the CS in the experimental group than in the control group. In contrast, somatomotor activity did not differ significantly between groups. Our results confirm the sensitivity of breathing to conditioning and indirectly support the hypothesis that learned feedforward processes may complement feedback pathways during postnatal maturation of respiratory control.     

Excitatory backward conditioning in an appetitive conditioned reinforcement preparation with rats

Four experiments were conducted to examine appetitive backward conditioning in a conditioned reinforcement preparation. In all experiments, off-line classical conditioning was conducted following lever-press training on two levers. Presentations of a sucrose solution by a liquid dipper served as an unconditioned stimulus (US) and two auditory stimuli served as conditioned stimuli (CSs); one was paired with the US in either a forward (Experiment 1a) or a backward (Experiments 1b, 2, and 3) relationship, and the other served as a control CS, which was not paired with the US. In testing, each lever-press response produced a presentation of one of the CSs instead of appetitive reinforcers. The response to a lever was facilitated, compared to the response to another lever, when the response produced the backward CS presentation as well as when it produced the forward CS presentation; that is, the backward CS served as an excitatory conditioned reinforcer.     

Pavlovian conditioning-specific increases of the Ca2+- and GTP-binding protein,calexcitin in identified Hermissenda visual cells

Hermissenda CNS, immunolabeled for the memory protein calexcitin showed significant immunostaining over background in the B-photoreceptor cells of the eye. The degree of staining correlated positively with the number of Pavlovian training events experienced by the animals and the degree of Pavlovian conditioning induced. The training regime consisted of exposing animals to light (conditioned stimulus, CS) paired with orbital rotation (unconditioned stimulus, US). In animals that exhibited the conditioned response, calexcitin immunolabeling was more intense than was found for naive (unconditioned) animals or animals given the CS and US in random sequence. Animals exposed to lead (maintained in 1.2 ppm lead acetate) at a dosage known to impair learning in children, showed reduced learning and less intense calexcitin staining whether the CS and US were paired or given randomly. However, the levels were still higher than that of naive animals. Immuno-electron microscopy indicated that the labeling was predominantly within calcium sequestering organelles such as the endoplasmic reticulum, and to lesser extent within mitochondria, and photopigments. The calexcitin density after a short-term memory (STM) regime was the same whether measured 5 minutes after conditioning (when STM was evidenced by foot contraction) or 90 minutes later when no recall was detected. The staining density was also similar to the levels found 5 minutes after long-term memory (LTM) conditioning. However, the LTM regime produced a greater calexcitin intensity at 90 minutes when the memory had been consolidated. This learning-specific increase in calexcitin is consistent with the previously implicated sequence of molecular events that are associated with progressively longer time domains of memory storage.     

Formation of new cortico-rubral synapses as a possible mechanism for classical conditioning mediated by the red nucleus in cat

1.) By extracellular and intracellular recordings of the red nucleus (RN) cell activity, we investigated enhancement of signaling effectiveness at the cortico-rubral synapses underlying the establishment of classical conditioning mediated by RN in the cat. The classical conditioning of forelimb flexion was produced by pairing the conditioned stimulus (CS) to the cerebral peduncle (CP) with the unconditioned stimulus (US) to the forelimb skin at an interval of 100 msec for about a week. 2.) The increased responsiveness of RN cells to the CS was correlated with acquisition of the conditioned forelimb flexion, i.e. RN cells responded to the CS with higher firing probability in the animals which received the paired conditioning than those in the animals which received the CS alone or pairing of the CS and the US at random intervals or those in the naive animals which did not receive any training. 3.) Monosynaptic excitation of RN cells in response to the single pulse to CP was most enhanced in the animals which received the paired conditioning. By contrast, response of RN cells, as well as the behavioral response, induced by stimulation of the cerebellar interpositus nucleus (IP) was not enhanced after the paired conditioning. The difference between the responses to the stimulation of CP and IP suggested that the primary site of neuronal change is the cortico-rubral synapses. 4.) In the animals that received the paired conditioning, the excitatory postsynaptic potentials (EPSPs) induced by stimulation of CP had fast-rising components superimposed on the normal slow-rising EPSPs. On the other hand, most of the CP-EPSPs recorded in the naive animals showed a slow time course. The slow time course of the CP-EPSPs has been attributed to the peripheral localization of the cortico-rubral synapses on the dendrites of RN cells. 5.) The electrotonic length of RN cells in the animals which received the paired conditioning was not shorter than that in the naive animals. Therefore, it was suggested that the appearance of the fast-rising component in the CP-EPSPs is cause by formation of the new cortico-rubral synapses on proximal portions of the soma-dendritic membrane of RN cells. 6.) Since it has been established that new synapses formed by collateral sprouting are retained for more than several months, the formation of new synaptic connections could underlie long-lasting behavioral modification.     

A simple and reliable test of olfactory learning and memory in mice

The present paper describes a quick and efficient method for assessing olfactory discrimination learning in mice. In training mice received trials in which one odor (CS+) was paired with sugar and another odor (CS-) was paired with no sugar. When the mice were subsequently placed in a chamber with CS+ odor at one end and CS- odor at the other, they spent more time digging in CS+ than in CS- odor. In Experiment 2 mice trained with this procedure and tested after 60 days also spent more time digging in CS+ than CS- in the test phase, indicating that this olfactory discrimination task is effective for assessing long-term memory. In addition to the outbred strain of CD1 mice used in Experiments 1 and 2, C57Bl/6NCr/BR and DBA/2NCr/BR mice used in Experiment 3 also acquired this learned odor discrimination. Moreover, Experiment 4 showed that DBA animals were capable of acquiring this odor discrimination after receiving only two training trials (one exposure each to CS+ and CS-) per day for 4 days.     

Training-Dependent Associative Learning Induced Neocortical Structural Plasticity: A Trace Eyeblink Conditioning Analysis

Studies utilizing general learning and memory tasks have suggested the importance of neocortical structural plasticity for memory consolidation. However, these learning tasks typically result in learning of multiple different tasks over several days of training, making it difficult to determine the synaptic time course mediating each learning event. The current study used trace-eyeblink conditioning to determine the time course for neocortical spine modification during learning. With eyeblink conditioning, subjects are presented with a neutral, conditioned stimulus (CS) paired with a salient, unconditioned stimulus (US) to elicit an unconditioned response (UR). With multiple CS-US pairings, subjects learn to associate the CS with the US and exhibit a conditioned response (CR) when presented with the CS. Trace conditioning is when there is a stimulus free interval between the CS and the US. Utilizing trace-eyeblink conditioning with whisker stimulation as the CS (whisker-trace-eyeblink: WTEB), previous findings have shown that primary somatosensory (barrel) cortex is required for both acquisition and retention of the trace-association. Additionally, prior findings demonstrated that WTEB acquisition results in an expansion of the cytochrome oxidase whisker representation and synaptic modification in layer IV of barrel cortex. To further explore these findings and determine the time course for neocortical learning-induced spine modification, the present study utilized WTEB conditioning to examine Golgi-Cox stained neurons in layer IV of barrel cortex. Findings from this study demonstrated a training-dependent spine proliferation in layer IV of barrel cortex during trace associative learning. Furthermore, findings from this study showing that filopodia-like spines exhibited a similar pattern to the overall spine density further suggests that reorganization of synaptic contacts set the foundation for learning-induced neocortical modifications through the different neocortical layers.     

Classical conditioning and retention in normal and mutantDrosophila melanogaster

Summary By changing the conditioned discrimination paradigm of Quinn et al. (1974) from an instrumental procedure to a classical (Pavlovian) one, we have demonstrated strong learning in type flies. About 150 flies were sequestered in a closed chamber and trained by explosing them sequentially to two odors in air currents. Flies received twelve electric shock pulses in the presence of the first odor (CS+) but not in the presence of the second odor (CS–). To test for conditioned avoidance responses, flies were transported to a Tmaze choice point, between converging currents of the two odors. Typically, 95% of trained flies avoided the shock-associated odor (CS+).Acquisition of learning was a function of the number of shock pulses received during CS+ presentation and was asymptotic within one training cycle. Conditioned avoidance increased with increasing shock intensity or odor concentration and was very resistant to extinction. Learning was best when CS+ presentations overlap shock (delay conditioning) and then decreased with increasing CS-US interstimulus intervals. Shocking flies immediately before CS+ presentation (backward conditioning) produced no learning. Nonassociative control procedures (CS Alone, US Alone and Explicitly Unpaired) produced slight decreases in avoidance responses, but these affected both odors equally and did not alter our associative learning index (A).Memory in wild-type flies decayed gradually over the first seven hours after training and still was present 24 h later. The mutantsamnesiac, rutabaga anddunce showed appreciable learning acquisition, but their memories decayed very rapidly during the first 30 min. After this, the rates of decay slowed sharply; conditioned avoidance still was measurable at least three hours after training.Abbreviations OCT 3-octanol - MCH 4-methylcyclohexanol - C-S Canton-Special - CS conditioned stimulus - US unconditioned stimulus     

A simple, single, trial-learning paradigm for conditioned increase in natural killer cell activity.

A change in natural killer (NK) cell activity can be conditioned with one trial learning when conditioned stimulus (CS) precedes the unconditioned stimulus (US). To avoid the problems associated with two reexposures in our earlier studies, we have developed a reliable and simple conditioning protocol utilizing the one trial learning and one reexposure to the odor CS. The conditioned change in NK cell activity was significantly different (P less than 0.05) from the control groups of mice. The paradigm is short and simple in that the conditioned change could be demonstrated within 3 days. We have also compared the effects of temporal association of CS and US on conditioned increase in NK cell activity. Forward conditioning (CS preceded the US) demonstrated a conditioned change, but the backward conditioning protocol did not. The paradigm provides a reliable approach to the study of mechanisms of the phenomenon of odor-NK conditioning.     

Does conditioned taste aversion learning in the pond snail Lymnaea stagnalis produce conditioned fear?

In conditioned taste aversion (CTA) training performed on the pond snail Lymnaea stagnalis, a stimulus (the conditional stimulus, CS; e.g., sucrose) that elicits a feeding response is paired with an aversive stimulus (the unconditional stimulus, US) that elicits the whole-body withdrawal response and inhibits feeding. After CTA training and memory formation, the CS no longer elicits feeding. We hypothesize that one reason for this result is that after CTA training the CS now elicits a fear response. Consistent with this hypothesis, we predict the CS will cause (1) the heart to skip a beat and (2) a significant change in the heart rate. Such changes are seen in mammalian preparations exposed to fearful stimuli. We found that in snails exhibiting long-term memory for one-trial CTA (i.e., good learners) the CS significantly increased the probability of a skipped heartbeat, but did not significantly change the heart rate. The probability of a skipped heartbeat was unaltered in control snails given backward conditioning (US followed by CS) or in snails that did not acquire associative learning (i.e., poor learners) after the one-trial CTA training. These results suggest that as a consequence of acquiring CTA, the CS evokes conditioned fear in the conditioned snails, as evidenced by a change in the nervous system control of cardiac activity.     

Properties of olfactory conditioning in the housefly, Musca domestica

The housefly, Musca domestica, was conditioned to odours using the proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response and the properties of conditioning were investigated. Among trials including forward pairing of the conditioned stimulus (CS) with the unconditioned stimulus (US), backward pairing and isolated presentations of CS and US, only forward pairing is effective on the acquisition of conditioning. Backward pairing combined with forward pairing does not influence the effectiveness of the forward pairing. CS given overlapping with a US presentation permits only weak conditioning. The acquisition of conditioning decreases with increase of the CS-US interval. In the differential conditioning situation to two odours, discriminative responses are observed. In the flies conditioned with one antenna, the conditioned response is elicited not only by stimulation of the antenna used for conditioning but also by stimulation of the antenna not used for conditioning, although the response using the former is higher than with the latter. The ability to be conditioned is reduced immediately after fastening on a clay bed and increases with time. Ability can also be improved by transection of the ventral nerve cord.     

Exposure to cold: aversive Pavlovian conditioning in individual Drosophila melanogaster

Abstract. Temperature changes can be especially threatening for ectotherms, such as Drosophila melanogaster (Diptera: Drosophilidea Meigen, 1830 ), and in this study we tested whether flies can associate olfactory stimuli with a sudden drop in temperature. Such Pavlovian conditioning would allow them to make appropriate behavioural and/or physiological responses in the future. We found that exposing individual flies to one of two odours in the presence of a sudden drop in temperature resulted in Pavlovian conditioning with flies subsequently avoiding the odour paired with cold. The characteristics of Pavlovian conditioning in flies were comparable to those observed for mammalian species. Specifically, the strength of conditioning increased with increasing intensity of the cold and decreased as the time interval between the olfactory stimulus (CS) and cold (US) was lengthened. Finally, the order in which CS and US were presented affected the strength of conditioning. Learning was observed when the CS preceded US and when the US immediately preceded the CS, but not when the CS preceded the US by 30 s or more. These results provide further evidence for learning in individual flies, and confirm that Pavlovian conditioning is a general mechanism used by organisms to obtain information about their environment.     

Light-induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae

During classical conditioning, a positive or negative value is assigned to a previously neutral stimulus, thereby changing its significance for behavior. If an odor is associated with a negative stimulus, it can become repulsive. Conversely, an odor associated with a reward can become attractive. By using Drosophila larvae as a model system with minimal brain complexity, we address the question of which neurons attribute these values to odor stimuli. In insects, dopaminergic neurons are required for aversive learning, whereas octopaminergic neurons are necessary and sufficient for appetitive learning. However, it remains unclear whether two independent neuronal populations are sufficient to mediate such antagonistic values. We report the use of transgenically expressed channelrhodopsin-2, a light-activated cation channel, as a tool for optophysiological stimulation of genetically defined neuronal populations in Drosophila larvae. We demonstrate that distinct neuronal populations can be activated simply by illuminating the animals with blue light. Light-induced activation of dopaminergic neurons paired with an odor stimulus induces aversive memory formation, whereas activation of octopaminergic/tyraminergic neurons induces appetitive memory formation. These findings demonstrate that antagonistic modulatory subsystems are sufficient to substitute for aversive and appetitive reinforcement during classical conditioning.     

Genotypic Influence on Aversive Conditioning in Honeybees,Using a Novel Thermal Reinforcement Procedure

In Pavlovian conditioning, animals learn to associate initially neutral stimuli with positive or negative outcomes, leading to appetitive and aversive learning respectively. The honeybee (Apis mellifera) is a prominent invertebrate model for studying both versions of olfactory learning and for unraveling the influence of genotype. As a queen bee mates with about 15 males, her worker offspring belong to as many, genetically-different patrilines. While the genetic dependency of appetitive learning is well established in bees, it is not the case for aversive learning, as a robust protocol was only developed recently. In the original conditioning of the sting extension response (SER), bees learn to associate an odor (conditioned stimulus - CS) with an electric shock (unconditioned stimulus - US). This US is however not a natural stimulus for bees, which may represent a potential caveat for dissecting the genetics underlying aversive learning. We thus first tested heat as a potential new US for SER conditioning. We show that thermal stimulation of several sensory structures on the bee’s body triggers the SER, in a temperature-dependent manner. Moreover, heat applied to the antennae, mouthparts or legs is an efficient US for SER conditioning. Then, using microsatellite analysis, we analyzed heat sensitivity and aversive learning performances in ten worker patrilines issued from a naturally inseminated queen. We demonstrate a strong influence of genotype on aversive learning, possibly indicating the existence of a genetic determinism of this capacity. Such determinism could be instrumental for efficient task partitioning within the hive.     

Pavlov's cockroach: classical conditioning of salivation in an insect

Secretion of saliva to aid swallowing and digestion is an important physiological function found in many vertebrates and invertebrates. Pavlov reported classical conditioning of salivation in dogs a century ago. Conditioning of salivation, however, has been so far reported only in dogs and humans, and its underlying neural mechanisms remain elusive because of the complexity of the mammalian brain. We previously reported that, in cockroaches Periplaneta americana, salivary neurons that control salivation exhibited increased responses to an odor after conditioning trials in which the odor was paired with sucrose solution. However, no direct evidence of conditioning of salivation was obtained. In this study, we investigated the effects of conditioning trials on the level of salivation. Untrained cockroaches exhibited salivary responses to sucrose solution applied to the mouth but not to peppermint or vanilla odor applied to an antenna. After differential conditioning trials in which an odor was paired with sucrose solution and another odor was presented without pairing with sucrose solution, sucrose-associated odor induced an increase in the level of salivation, but the odor presented alone did not. The conditioning effect lasted for one day after conditioning trials. This study demonstrates, for the first time, classical conditioning of salivation in species other than dogs and humans, thereby providing the first evidence of sophisticated neural control of autonomic function in insects. The results provide a useful model system for studying cellular basis of conditioning of salivation in the simpler nervous system of insects.     

Critical Period of Memory Enhancement during Taste Avoidance Conditioning in Lymnaea stagnalis

The present study investigated the optimal training procedure leading to long-lasting taste avoidance behavior in Lymnaea. A training procedure comprising 5 repeated pairings of a conditional stimulus (CS, sucrose), with an unconditional stimulus (US, a tactile stimulation to the animal’s head), over a 4-day period resulted in an enhanced memory formation than 10 CS-US repeated pairings over a 2-day period or 20 CS-US repeated pairings on a single day. Backward conditioning (US-CS) pairings did not result in conditioning. Thus, this taste avoidance conditioning was CS-US pairing specific. Food avoidance behavior was not observed following training, however, if snails were immediately subjected to a cold-block (4°C for 10 min). It was critical that the cold-block be applied within 10 min to block long-term memory (LTM) formation. Further, exposure to the cold-block 180 min after training also blocked both STM and LTM formation. The effects of the cold-block on subsequent learning and memory formation were also examined. We found no long lasting effects of the cold-block on subsequent memory formation. If protein kinase C was activated before the conditioning paradigm, snails could still acquire STM despite exposure to the cold-block.