Second-order conditioning in Drosophila

Associative conditioning in Drosophila melanogaster has been well documented for several decades. However, most studies report only simple associations of conditioned stimuli (CS, e.g., odor) with unconditioned stimuli (US, e.g., electric shock) to measure learning or establish memory. Here we describe a straightforward second-order conditioning (SOC) protocol that further demonstrates the flexibility of fly behavior. In SOC, a previously conditioned stimulus (CS1) is used as reinforcement for a second conditioned stimulus (CS2) in associative learning. This higher-order context presents an opportunity for reassessing the roles of known learning and memory genes and neuronal networks in a new behavioral paradigm.     

Evaluation and development of a connectionist theory of configural learning

A configural theory of associative learning is described that is based on the assumption that conditioning results in associations between the unconditioned stimulus and a representation of the entire pattern of stimulation that was present prior to its delivery. Configural theory was formulated originally to account for generalization and discrimination in Pavlovian conditioning. The first part of the article demonstrates how this theory can be used to explain results from studies of overshadowing, blocking, summation, and discrimination learning. The second part of the article shows how the theory can be developed to explain a broader range of phenomena, including mediated conditioning, reinforcer devaluation effects, the differential outcomes effect, acquired equivalence, sensory preconditioning, and structural discriminations.     

Activation of MAPK is necessary for long-term memory consolidation following food-reward conditioning

Although an important role for the mitogen-activated protein kinase (MAPK) has been established for memory consolidation in a variety of learning paradigms, it is not known if this pathway is also involved in appetitive classical conditioning. We address this question by using a single-trial food-reward conditioning paradigm in the freshwater snail Lymnaea stagnalis. This learning paradigm induces protein synthesis-dependent long-term memory formation. Inhibition of MAPK phosphorylation blocked long-term memory consolidation without affecting the sensory and motor abilities of the snails. Thirty minutes after conditioning, levels of MAPK phosphorylation were increased in extracts from the buccal and cerebral ganglia. These ganglia are involved in the generation, modulation, and plasticity of the feeding behavior. We also detected an increase in levels of MAPK phosphorylation in the peripheral tissue around the mouth of the snails where chemoreceptors are located. Although an increase in MAPK phosphorylation was shown to be essential for food-reward conditioning, it was also detected in snails that were exposed to the conditioned stimulus (CS) or the unconditioned stimulus (US) alone, suggesting that phosphorylation of MAPK is necessary but not sufficient for learning to occur.     

Sensory preconditioning and UCS inflation in human 'fear' conditioning

This study describes a human electrodermal conditioning experiment involving processes of sensory preconditioning and UCS inflation. In stage 1 of the experiment Ss received six presentations of a CS+ paired with an innocuous 65 dB tone (UCS) and six presentations of an unpaired CS-. In stage 2, Ss in the experimental group had the aversiveness of the UCS inflated as the intensity of the 65 dB tone was increased to 115 dB. In stage 3, Ss were given test presentations of CS+ and CS-. A differential CR to CS+ was found only in stage 3 of the experiment and only in Ss who had experienced the UCS inflation procedure. These results suggest that (i) sensory preconditioning had occurred in stage 1 despite the failure to observe a differential CR in this stage, and (ii) the differential CR observed in stage 3 was mediated by an internal representation of the UCS whose aversiveness had been inflated in stage 2. As well as confirming that processes of sensory preconditioning and UCS inflation can be observed in human as well as animal Ss, these findings have important implications for contemporary conditioning models of clinical fears. In particular, they suggest that a contemporary conditioning model of acquired fears is not bound by the need to discover contiguous stimulus-trauma experiences in the histories of clinical phobias since, in humans at least, processes of stimulus association and UCS revaluation appear to be relatively independent.     

Blocking and sensory preconditioning effects in morphine analgesic tolerance: Support for a pavlovian conditioning model of drug tolerance

Two experiments on rats tested predictions of a Pavlovian conditioning model of drug tolerance which holds that tolerance is the result of compensatory conditioned responses, developed to environmental stimuli accompanying the drug administrations, which attenuate the direct effects of the drug. In Experiment I, the acquisition of tolerance-modulating properties by the tone component of a tone-light compound stimulus which accompanied morphine administrations was reduced by prior light-morphine pairings (blocking). In Experiment II, a tone stimulus acquired tolerance-modulating properties through prior pairings with a light stimulus which later accompanied morphine administrations (sensory preconditioning). These findings are uniquely predicted by the Pavlovian conditioning model of drug tolerance and are incompatible with traditional theories which assign no role to environmental stimuli present at the time of drug administration.     

Learning with arbitrary versus ecological conditioned stimuli: Evidence from sexual conditioning

Laboratory investigations of Pavlovian conditioning typically involve the association of an arbitrary conditioned stimulus (CS) with an unconditioned stimulus (US) that has no inherent relation to the CS. However, arbitrary CSs are unlikely to become conditioned outside the laboratory, because they do not occur often enough with the US to result in an association. Learning under natural circumstances is likely only if the CS has a preexisting relation to the US. Recent studies of sexual conditioning have shown that in contrast to an arbitrary CS, an ecologically relevant CS is resistant to blocking, extinction, and increases in the CS-US interval and results in sensitized responding and stronger second-order conditioning. Although the mechanisms of these effects are not fully understood, these findings have shown that signature learning phenomena are significantly altered when the kinds of stimuli that are likely to become conditioned under natural circumstances are used. The implications of these findings for an ecological approach to the study of learning are discussed.     

Pavlovian conditioning of Hermissenda: current cellular, molecular, and circuit perspectives

The less-complex central nervous system of many invertebrates make them attractive for not only the molecular analysis of the associative learning and memory, but also in determining how neural circuits are modified by learning to generate changes in behavior. The nudibranch mollusk Hermissenda crassicornis is a preparation that has contributed to an understanding of cellular and molecular mechanisms of Pavlovian conditioning. Identified neurons in the conditioned stimulus (CS) pathway have been studied in detail using biophysical, biochemical, and molecular techniques. These studies have resulted in the identification and characterization of specific membrane conductances contributing to enhanced excitability and synaptic facilitation in the CS pathway of conditioned animals. Second-messenger systems activated by the CS and US have been examined, and proteins that are regulated by one-trial and multi-trial Pavlovian conditioning have been identified in the CS pathway. The recent progress that has been made in the identification of the neural circuitry supporting the unconditioned response (UR) and conditioned response (CR) now provides for the opportunity to understand how Pavlovian conditioning is expressed in behavior.     

Contextual Pavlovian conditioning in the crab Chasmagnathus

In contextual conditioning, a complex pattern of information is processed to associate the characteristics of a particular place with incentive or aversive reinforcements. This type of learning has been widely studied in mammals, but studies of other taxa are scarce. The context-signal memory (CSM) paradigm of the crab Chasmagnathus has been extensively used as a model of learning and memory. Although initially interpreted as habituation, some characteristics of contextual conditioning have been described. However, no anticipatory response has been detected for animals exposed to the training context. Thus, CSM could be interpreted either as an associative habituation or as contextual conditioning that occurs without a context-evoked anticipatory response. Here, we describe a training protocol developed for contextual Pavlovian conditioning (CPC). For each training trial, the context (conditioned stimulus, CS) was discretely presented and finished together with the unconditioned stimulus (US). In agreement with the CSM paradigm, a robust freezing response was acquired during the 15 training trials, and clear retention was found when tested with the US presentation after short (2 and 4 h) and long (1–4 days) delays. This CPC memory showed forward but not simultaneous presentation conditioning and was context specific and protein synthesis dependent. Additionally, a weak CPC memory was enhanced during consolidation. One day after training, CPC was extinguished by repeated CS presentation, while one presentation induced a memory labilisation–reconsolidation process. Finally, we found an anticipatory conditioned response (CR) during the CS presentation for both short-term (4 h) and long-term memory (24 h). These findings support the conditioning nature of the new paradigm.     

Post-training amphetamine administration enhances memory consolidation in appetitive Pavlovian conditioning: Implications for drug addiction

It has been suggested that some of the addictive potential of psychostimulant drugs of abuse such as amphetamine may result from their ability to enhance memory for drug-related experiences through actions on memory consolidation. This experiment examined whether amphetamine can specifically enhance consolidation of memory for a Pavlovian association between a neutral conditioned stimulus (CS-a light) and a rewarding unconditioned stimulus (US-food), as Pavlovian conditioning of this sort plays a major role in drug addiction. Male Long-Evans rats were given six training sessions consisting of 8 CS presentations followed by delivery of the food into a recessed food cup. After the 1st, 3rd, and 5th session, rats received subcutaneous injections of amphetamine (1.0 or 2.0 mg/kg) or saline vehicle immediately following training. Conditioned responding was assessed using the percentage of time rats spent in the food cup during the CS relative to a pre-CS baseline period. Both amphetamine-treated groups showed significantly more selective conditioned responding than saline controls. In a control experiment, there were no differences among groups given saline, 1.0 or 2.0 mg/kg amphetamine 2 h post-training, suggesting that immediate post-training amphetamine enhanced performance specifically through actions on memory consolidation rather than through non-mnemonic processes. This procedure modeled Pavlovian learning involved in drug addiction, in which the emotional valence of a drug reward is transferred to neutral drug-predictive stimuli such as drug paraphernalia. These data suggest that amphetamine may contribute to its addictive potential through actions specifically on memory consolidation.     

An analysis of second-order autoshaping

Three mechanisms can explain second-order conditioning: (1) The second-order conditioned stimulus (CS2) could activate a representation of the first-order conditioned stimulus (CS1), thereby provoking the conditioned response (CR); The CS2 could enter into an excitatory association with either (2) the representation governing the CR, or (3) with a representation of the reinforcer evoked by the CS1. A series of experiments using second-order autoshaping with birds was performed to examine these possibilities. Following second-order autoshaping, birds’ responding to the CS2 was found to be unaffected by extinction of the CS1, a result interpreted as showing mechanism (1) to be unimportant and implicating either one or both of the other mechanisms. The CS2 was also shown to provide the birds with specific sensory information about the reinforcer, a pattern of results uniquely predicted by mechanism (3). Implications for the understanding of second-order conditioning and for the SOP model (Wagner, 1981) of associative learning are discussed.     

Associative representational plasticity in the auditory cortex: a synthesis of two disciplines

Historically, sensory systems have been largely ignored as potential loci of information storage in the neurobiology of learning and memory. They continued to be relegated to the role of "sensory analyzers" despite consistent findings of associatively induced enhancement of responses in primary sensory cortices to behaviorally important signal stimuli, such as conditioned stimuli (CS), during classical conditioning. This disregard may have been promoted by the fact that the brain was interrogated using only one or two stimuli, e.g., a CS(+) sometimes with a CS(-), providing little insight into the specificity of neural plasticity. This review describes a novel approach that synthesizes the basic experimental designs of the experimental psychology of learning with that of sensory neurophysiology. By probing the brain with a large stimulus set before and after learning, this unified method has revealed that associative processes produce highly specific changes in the receptive fields of cells in the primary auditory cortex (A1). This associative representational plasticity (ARP) selectively facilitates responses to tonal CSs at the expense of other frequencies, producing tuning shifts toward and to the CS and expanded representation of CS frequencies in the tonotopic map of A1. ARPs have the major characteristics of associative memory: They are highly specific, discriminative, rapidly acquired, exhibit consolidation over hours and days, and can be retained indefinitely. Evidence to date suggests that ARPs encode the level of acquired behavioral importance of stimuli. The nucleus basalis cholinergic system is sufficient both for the induction of ARPs and the induction of specific auditory memory. Investigation of ARPs has attracted workers with diverse backgrounds, often resulting in behavioral approaches that yield data that are difficult to interpret. The advantages of studying associative representational plasticity are emphasized, as is the need for greater behavioral sophistication.     

Electrolytic lesions of the dorsal hippocampus disrupt renewal of conditional fear after extinction

There is a growing body of evidence that the hippocampus is critical for context-dependent memory retrieval. In the present study, we used Pavlovian fear conditioning in rats to examine the role of the dorsal hippocampus (DH) in the context-specific expression of fear memory after extinction (i.e., renewal). Pre-training electrolytic lesions of the DH blunted the expression of conditional freezing to an auditory conditional stimulus (CS), but did not affect the acquisition of extinction to that CS. In contrast, DH lesions impaired the context-specific expression of extinction, eliminating the renewal of fear normally observed to a CS presented outside of the extinction context. Post-extinction DH lesions also eliminated the context dependence of fear extinction. These results are consistent with those using pharmacological inactivation of the DH and suggest that the DH is required for using contextual stimuli to regulate the expression of fear to a Pavlovian CS after extinction.     

Associative learning acquisition and retention depends on developmental stage in Lymnaea stagnalis

Associative learning dependent on visual and vestibular sensory neurons and the underlying cellular mechanisms have been well characterized in Hermissenda but not yet in Lymnaea. Three days of conditioning with paired presentations of a light flash (conditional stimulus: CS) and orbital rotation (unconditional stimulus: UCS) in intact Lymnaea stagnalis results in a whole-body withdrawal response (WBWR) to the CS. In the current study, we examined the optimal stimulus conditions for associative learning, including developmental stage, number of stimuli, interstimulus interval, and intertrial interval. Animals with a shell length longer than 18 mm (sexually mature) acquired and retained the associative memory, while younger ones having a shell length shorter than 15 mm acquired but did not retain the memory to the following day. For mature animals, 10 paired presentations of the CS and UCS presented every 2 min were sufficient for the induction of a WBWR to the CS. Furthermore, animals conditioned with the UCS presented simultaneously with the last 2 s of the CS also exhibited a significant WBWR in response to the CS. Blind animals did not acquire the associative memory, suggesting that ocular photoreceptors, and not dermal photoreceptors, detected the CS. These results show that maturity was key to retention of associative learning.     

Pavlovian second-order conditioned analgesia

Three experiments with rat subjects assessed conditioned analgesia in a Pavlovian second-order conditioning procedure by using inhibition of responding to thermal stimulation as an index of pain sensitivity. In Experiment 1, rats receiving second-order conditioning showed longer response latencies during a test of pain sensitivity in the presence of the second-order conditioned stimulus (CS) than rats receiving appropriate control procedures. Experiment 2 found that extinction of the first-order CS had no effect on established second-order conditioned analgesia. Experiment 3 evaluated the effects of post second-order conditioning pairings of morphine and the shock unconditioned stimulus (US). Rats receiving paired morphine-shock presentations showed significantly shorter response latencies during a hot-plate test of pain sensitivity in the presence of the second-order CS than did groups of rats receiving various control procedures; second-order analgesia was attenuated. These data extend the associative account of conditioned analgesia to second-order conditioning situations and are discussed in terms of the mediation of both first- and second-order analgesia by an association between the CS and a representation or expectancy of the US, which may directly activate endogenous pain inhibition systems.     

Learned fear of "unseen" faces after Pavlovian, observational, and instructed fear

This study compared fear learning acquired through direct experience (Pavlovian conditioning) and fear learning acquired without direct experience via either observation or verbal instruction. We examined whether these three types of learning yielded differential responses to conditioned stimuli (CS+) that were presented unmasked (available to explicit awareness) or masked (not available to explicit awareness). In the Pavlovian group, the CS+ was paired with a mild shock, whereas the observational-learning group learned through observing the emotional expression of a confederate receiving shocks paired with the CS+. The instructed-learning group was told that the CS+ predicted a shock. The three groups demonstrated similar levels of learning as measured by the skin conductance response to unmasked stimuli. As in previous studies, participants also displayed a significant learning response to masked stimuli following Pavlovian conditioning. However, whereas the observational-learning group also showed this effect, the instructed-learning group did not.     

Olfactory fear conditioning induces field potential potentiation in rat olfactory cortex and amygdala

The widely used Pavlovian fear-conditioning paradigms used for studying the neurobiology of learning and memory have mainly used auditory cues as conditioned stimuli (CS). The present work assessed the neural network involved in olfactory fear conditioning, using olfactory bulb stimulation-induced field potential signal (EFP) as a marker of plasticity in the olfactory pathway. Training consisted of a single training session including six pairings of an odor CS with a mild foot-shock unconditioned stimulus (US). Twenty-four hours later, the animals were tested for retention of the CS as assessed by the amount of freezing exhibited in the presence of the learned odor. Behavioral data showed that trained animals exhibited a significantly higher level of freezing in response to the CS than control animals. In the same animals, EFPs were recorded in parallel in the anterior piriform cortex (aPC), posterior piriform cortex (pPC), cortical nucleus of the amygdala (CoA), and basolateral nucleus of the amygdala (BLA) following electrical stimulation of the olfactory bulb. Specifically, EFPs recorded before (baseline) and after (during the retention test) training revealed that trained animals exhibited a lasting increase (present before and during presentation of the CS) in EFP amplitude in CoA, which is the first amygdaloid target of olfactory information. In addition, a transient increase was observed in pPC and BLA during presentation of the CS. These data indicate that the olfactory and auditory fear-conditioning neural networks have both similarities and differences, and suggest that the fear-related behaviors in each sensory system may have at least some distinct characteristics.     

UCS revaluation and conditioning models of acquired fears

Recent developments in the study of Pavlovian conditioning in humans have suggested that the strength of a conditioned response (CR) is affected not only by associative processes that link the conditioned stimulus (CS) with the unconditioned stimulus (UCS), but also by processes which lead the individual to revalue the UCS itself. These latter processes can frequently lead to dramatic changes in CR strength independently of any experience with the CS-UCS contingency. This paper discusses a number of ways in which UCS revaluation can be effected in human subjects, and discusses their implications for conditioning models of acquired fears.     

Direct and continuous measurement of relational learning in human pavlovian conditioning

Three experiments were conducted employing a continuous measure of conditional stimulus/unconditional stimulus (CS/US) contingencies as perceived by the subject (i.e., subjective contingency or SC). It is argued that direct measurement of relational learning, as indexed by SC, can lead to a better understanding of Pavlovian conditioning processes. The first two experiments applied this approach to a methodologic controversy, raising the debate from a procedure-based argument to testing what the subject actually learns about CS/US relationships. While the issue was not resolved, testable hypotheses for future research were generated from the data. The third experiment contrasted the contingency stimulus-stimulus (S-S) account of Pavlovian conditioning with an earlier stimulus-response (S-R) continguity-reinforcement account. In this experiment, both SC and skin resistance were measured. Evidence for the existence of both cognitive-propositional and response-learning processes in conditioning was obtained.     

Hippocampal regulation of context-dependent neuronal activity in the lateral amygdala

Pavlovian fear conditioning is a robust and enduring form of emotional learning that provides an ideal model system for studying contextual regulation of memory retrieval. After extinction the expression of fear conditional responses (CRs) is context-specific: A conditional stimulus (CS) elicits greater conditional responding outside compared with inside the extinction context. Dorsal hippocampal inactivation with muscimol attenuates context-specific CR expression. We have previously shown that CS-elicited spike firing in the lateral nucleus of the amygdala is context-specific after extinction. The present study examines whether dorsal hippocampal inactivation with muscimol disrupts context-specific firing in the lateral amygdala. We conditioned rats to two separate auditory CSs and then extinguished each CS in separate and distinct contexts. Thereafter, single-unit activity and conditional freezing were tested to one CS in both extinction contexts after saline or muscimol infusion into the dorsal hippocampus. After saline infusion, rats froze more to the CS when it was presented outside of its extinction context, but froze equally in both contexts after muscimol infusion. In parallel with the behavior, lateral nucleus neurons exhibited context-dependent firing to extinguished CSs, and hippocampal inactivation disrupted this activity pattern. These data reveal a novel role for the hippocampus in regulating the context-specific firing of lateral amygdala neurons after fear memory extinction.     

Undernutrition during the brain growth period of the rat significantly delays the development of processes mediating Pavlovian trace conditioning

Pavlovian trace conditioning procedures allow one to assess the ability of animals to associate events that are temporally separated because the conditioned stimulus terminates prior to the occurrence of the unconditioned stimulus. We report that undernutrition during Postnatal Days 2-18 significantly delays the development of trace conditioning to a visual stimulus. Previously undernourished 20-day-old rats conditioned when no temporal interval separated the termination of the CS and US onset (0-s trace interval). However, it was not until the undernourished pups were 30 days old that they conditioned when the trace interval was 10 or 30 s. In contrast, control pups only 20 days old were able to condition when a 10-s trace interval separated the CS and US events, and 25-day-old control pups conditioned when the interval was either 10 or 30 s. These results suggest that undernutrition delays the development of processes that enable the rat to sustain a representation of a visual CS during the trace interval.