Measuring Pavlovian appetitive conditioning in humans with the postauricular reflex

Despite its evolutionary and clinical significance, appetitive conditioning has been rarely investigated in humans. It has been proposed that this discrepancy might stem from the difficulty in finding suitable appetitive stimuli that elicit strong physiological responses. However, this might also be due to a possible lack of sensitivity of the psychophysiological measures commonly used to index human appetitive conditioning. Here, we investigated whether the postauricular reflex—a vestigial muscle microreflex that is potentiated by pleasant stimuli relative to neutral and unpleasant stimuli—may provide a valid psychophysiological indicator of appetitive conditioning in humans. To this end, we used a delay differential appetitive conditioning procedure, in which a neutral stimulus was contingently paired with a pleasant odor (CS+), while another neutral stimulus was not associated with any odor (CS?). We measured the postauricular reflex, the startle eyeblink reflex, and skin conductance response (SCR) as learning indices. Taken together, our results indicate that the postauricular reflex was potentiated in response to the CS+ compared with the CS?, whereas this potentiation extinguished when the pleasant odor was no longer delivered. In contrast, we found no evidence for startle eyeblink reflex attenuation in response to the CS+ relative to the CS?, and no effect of appetitive conditioning was observed on SCR. These findings suggest that the postauricular reflex is a sensitive measure of human appetitive conditioning and constitutes a valuable tool for further shedding light on the basic mechanisms underlying emotional learning in humans.     

Measuring the conditioned response: A comparison of pupillometry,skin conductance,and startle electromyography

In human fear conditioning studies, different physiological readouts can be used to track conditioned responding during fear learning. Commonly employed readouts such as skin conductance responses (SCR) or startle responses have in recent years been complemented by pupillary readouts, but to date it is unknown how pupillary readouts relate to other measures of the conditioned response. To examine differences and communalities among pupil responses, SCR, and startle responses, we simultaneously recorded pupil diameter, skin conductance, and startle electromyography in 47 healthy subjects during fear acquisition, extinction, and a recall test on 2 consecutive days. The different measures correlated only weakly, displaying most prominent differences in their response patterns during fear acquisition. Whereas SCR and startle responses habituated, pupillary measures did not. Instead, they increased in response to fear conditioned stimuli and most closely followed ratings of unconditioned stimulus (US) expectancy. Moreover, we observed that startle‐induced pupil responses showed stimulus discrimination during fear acquisition, suggesting a fear potentiation of the auditory pupil reflex. We conclude that different physiological outcome measures of the conditioned response inform about different cognitive‐affective processes during fear learning, with pupil responses being least affected by physiological habituation and most closely following US expectancy.     

A demonstration of classical conditioning of the human eyeblink to an olfactory stimulus.

While acquisition of the eyeblink conditioned response to a variety of stimuli has been widely studied, it has yet to be established that humans will demonstrate a conditioned response to an olfactory stimulus. In this study we present data to show that humans will demonstrate a classically conditioned eyeblink response to an olfactory stimulus. Ten participants were tested in a delay conditioning procedure with an olfactory stimulus presented in a heated, humidified stream of air via an olfactometer, allowing the precise control over stimulus duration necessary for delay conditioning. Trials on which odor alone was presented were administered to four additional participants. Establishing that humans will demonstrate an eyeblink conditioned response to an olfactory stimulus will allow further exploration of the pathways involved in classical conditioning and associative learning, as well as an analysis of conditioning pathways across sensory modalities.     

Pupil dilation indicates the coding of past prediction errors: Evidence for attentional learning theory

The attentional learning theory of Pearce and Hall ( 1980 ) predicts more attention to uncertain cues that have caused a high prediction error in the past. We examined how the cue‐elicited pupil dilation during associative learning was linked to such error‐driven attentional processes. In three experiments, participants were trained to acquire associations between different cues and their appetitive (Experiment 1), motor (Experiment 2), or aversive (Experiment 3) outcomes. All experiments were designed to examine differences in the processing of continuously reinforced cues (consistently followed by the outcome) versus partially reinforced, uncertain cues (randomly followed by the outcome). We measured the pupil dilation elicited by the cues in anticipation of the outcome and analyzed how this conditioned pupil response changed over the course of learning. In all experiments, changes in pupil size complied with the same basic pattern: During early learning, consistently reinforced cues elicited greater pupil dilation than uncertain, randomly reinforced cues, but this effect gradually reversed to yield a greater pupil dilation for uncertain cues toward the end of learning. The pattern of data accords with the changes in prediction error and error‐driven attention formalized by the Pearce‐Hall theory.     

A Comparison of Two Model Systems of Associative Learning: Heart Rate and Eyeblink Conditioning in the Rabbit

In four classical conditioning experiments heart period and eyeblink responses were assessed concomitantly. The conditioned stimuli (CSs) were tones and the unconditioned stimulus (UCS) was a brief paraorbital electric shock. Using a 0.5-s duration CS, bradycardiac conditioned responses that consisted of a 4–5 ms change from pre-CS baseline occurred within 10–20 CS-UCS presentations. However, eyeblink conditioned responses began to occur only after 100–150 CS–UCS presentations. A 1.0-s CS duration resulted in bradycardiac conditioned responses of 15–30 ms change from pre-CS baseline, which again reached asymptote within 10–20 trials. Using a 4-s CS duration, in a differential classical conditioning paradigm, heart period discrimination between a reinforced CS+ and a nonreinforced CS? occurred within 10 trials; asymptotic performance of the heart period conditioned response occurred within 15 CS-UCS presentations and consisted of a bradycardiac response of 40–50 ms for the 12th interbeat interval following tone onset. These data thus indicate that these two model systems of mammalian learning (based on heart period and eyeblink responses) show quite different acquisition functions. It is also significant that heart rate slowing always accompanied the eyeblink conditioned responses, even though increases in general electromyographic activity as well as eyeblink conditioned responses were simultaneously observed during CS presentation.     

A pupil size response model to assess fear learning

During fear conditioning, pupil size responses dissociate between conditioned stimuli that are contingently paired (CS+) with an aversive unconditioned stimulus, and those that are unpaired (CS‐). Current approaches to assess fear learning from pupil responses rely on ad hoc specifications. Here, we sought to develop a psychophysiological model (PsPM) in which pupil responses are characterized by response functions within the framework of a linear time‐invariant system. This PsPM can be written as a general linear model, which is inverted to yield amplitude estimates of the eliciting process in the central nervous system. We first characterized fear‐conditioned pupil size responses based on an experiment with auditory CS. PsPM‐based parameter estimates distinguished CS+/CS‐ better than, or on par with, two commonly used methods (peak scoring, area under the curve). We validated this PsPM in four independent experiments with auditory, visual, and somatosensory CS, as well as short (3.5 s) and medium (6 s) CS/US intervals. Overall, the new PsPM provided equal or decisively better differentiation of CS+/CS‐ than the two alternative methods and was never decisively worse. We further compared pupil responses with concurrently measured skin conductance and heart period responses. Finally, we used our previously developed luminance‐related pupil responses to infer the timing of the likely neural input into the pupillary system. Overall, we establish a new PsPM to assess fear conditioning based on pupil responses. The model has a potential to provide higher statistical sensitivity, can be applied to other conditioning paradigms in humans, and may be easily extended to nonhuman mammals.     

Event‐related potentials to threat of predictable and unpredictable shock

Cognitive affective neuroscience tasks that are straightforward to administer, measure key constructs of interest, and can be used in different lab settings and with multiple psychophysiological methods can lead to a more complete understanding of experimental effects. The no‐threat, predictable threat, unpredictable threat (NPU‐threat) task assesses constructs of interest to both clinical and basic affective science literatures, is relatively brief to administer, and has been used across labs with a number of different measurements (e.g., startle eyeblink, fMRI, corrugator response, subjective ratings). ERPs provide another means of assessing neurobiological reactivity during the NPU‐threat task, but to date such measures have been underutilized. That is, no study has yet evaluated cue‐elicited ERPs in the NPU‐threat task. Here, cue‐elicited ERPs were assessed in 78 participants who completed a version of the NPU‐threat task previously shown to reliably moderate startle eyeblink amplitudes. Results showed larger P2 amplitudes for unpredictable versus predictable trials, increased P3s and late positive potentials for threatening versus no‐threat trials, as well as larger stimulus preceding negativities for threatening versus no‐threat trials (driven primarily by predictable threat cues). In line with prior work, we observed enhanced startle eyeblink for threatening versus no‐threat trials and for unpredictable compared to predictable threat interstimulus intervals. In addition, the probe‐elicited P3 was suppressed for predictable and unpredictable compared to no‐threat trials. Therefore, cue‐elicited ERPs, which can be recorded alongside other measures in the NPU‐threat task (e.g., startle), may provide useful indices of temporally distinct stages of predictable and unpredictable threat processing.     

Modeling startle eyeblink electromyogram to assess fear learning

Pavlovian fear conditioning is widely used as a laboratory model of associative learning in human and nonhuman species. In this model, an organism is trained to predict an aversive unconditioned stimulus from initially neutral events (conditioned stimuli, CS). In humans, fear memory is typically measured via conditioned autonomic responses or fear‐potentiated startle. For the latter, various analysis approaches have been developed, but a systematic comparison of competing methodologies is lacking. Here, we investigate the suitability of a model‐based approach to startle eyeblink analysis for assessment of fear memory, and compare this to extant analysis strategies. First, we build a psychophysiological model (PsPM) on a generic startle response. Then, we optimize and validate this PsPM on three independent fear‐conditioning data sets. We demonstrate that our model can robustly distinguish aversive (CS+) from nonaversive stimuli (CS‐, i.e., has high predictive validity). Importantly, our model‐based approach captures fear‐potentiated startle during fear retention as well as fear acquisition. Our results establish a PsPM‐based approach to assessment of fear‐potentiated startle, and qualify previous peak‐scoring methods. Our proposed model represents a generic startle response and can potentially be used beyond fear conditioning, for example, to quantify affective startle modulation or prepulse inhibition of the acoustic startle response.     

Individual Differences in Autonomic Response: Conditioned Association or Conditioned Fear?

Cluster analysis was used to define three groups of subjects whose conditioned heart rate response emphasized either acceleration, deceleration, or moderate deceleration. A subject pool (N = 148) was generated from four separate studies of differential classical conditioning in which colored slides served as conditioned stimuli (CSs) and the unconditioned stimulus (UCS) was aversive auditory noise. Both the statistically generated Accelerator and Decelerator groups responded with a significant late interval deceleration to CS+. However, only Accelerators also displayed a significant mid-interval heart rate increase to CS+. Conditioned electrodermal responses were significantly more resistant to extinction for Accelerators than for the other two cluster groups. Accelerators also differed from Decelerators in that their affective judgments of the slide stimuli became less positively valent and less dominant from pre- to post-conditioning, effects not found for Decelerators. Moderate Decelerators produced mixed results across the several psychophysiological and verbal measures of conditioning, resembling Accelerators in their verbal report change, and Decelerators in their rapid electrodermal extinction. It is suggested that cluster assignment may reflect subjects' pre-conditioning learning set. All successfully conditioned subjects appear to learn a relationship between stimuli such that the appearance of the CS+ prompts an anticipatory orienting response (late interval heart rate deceleration). Accelerators differ from the other two groups in that they also condition a defensive response which is associated with a change in emotional judgments of the stimuli and may represent covert preparation for avoidance. The relationships between defensive responding, avoidance, and the concept of fear are considered, and the problem of desynchrony in emotional change (i.e., the Moderate Decelerators) is addressed. Finally, parallels are drawn between results from classical conditioning and clinical studies of fear and phobia, and further suggestions are made for utilizing the conditioning paradigm in the study of emotion.     

Stimulus processing and associative learning in Wistar and WKHA rats

This study assessed basic learning and attention abilities in Wistar-Kyoto hyperactive (WKHA) rats using appetitive conditioning preparations. Two measures of conditioned responding to a visual stimulus, orienting behavior (rearing on the hind legs), and food cup behavior (placing the head inside the recessed food cup) were measured. In Experiment 1, simple conditioning, but not extinction, was impaired in WKHA rats compared with Wistar rats. In Experiment 2, nonreinforced presentations of the visual cue preceded the conditioning sessions. WKHA rats displayed less orienting behavior than Wistar rats but comparable levels of food cup behavior. These data suggest that WKHA rats exhibit specific abnormalities in attentional processing as well as in learning stimulus-reward relationships.     

Effects of age and awareness on eyeblink conditional discrimination learning

The present study aimed to assess the effects of aging and awareness on conditional discrimination learning within an eyeblink conditioning procedure by using a consecutive age-groups design (20-35 years, 36-50 years, 51-65 years, 66-80 years). Increasing age was associated with a decline in overall eyeblink conditioned response (CR) frequency and a deficit in conditional discrimination learning in the 2 older groups. Awareness of stimulus contingencies affected discrimination performance but not overall CR rates in younger subjects. Older subjects did not achieve eyeblink conditional discrimination learning, regardless of awareness. Discrimination performance correlated with measures of declarative memory. The pattern of results is discussed with respect to the involvement of hippocampal-cerebellar interactions and awareness in the mediation of age-related conditioning changes.     

Dorsal hippocampal involvement in appetitive trace conditioning and interval timing

Involvement of the dorsal hippocampus (DHPC) in acquisition of Pavlovian trace conditioning and interval timing was examined in an appetitive preparation in which presentations of one conditioned stimulus (CS) were immediately followed by food (delay conditioning), and presentations of another CS were followed by food 15 seconds after its termination (trace conditioning). DHPC lesions did not disrupt acquisition of trace conditioning, but they selectively affected the distribution of conditioned responding over the course of the trace CS in the early stage of acquisition. In addition, lesions disrupted accuracy of timing the conditioned response (CR) for both delay and trace CSs: The control subjects showed maximum CR at the time of food delivery, but in the DHPC-lesioned subjects, the maximum CR was at an earlier time point. This timing deficit did not seem to be due to impulsive responding or deficits in response inhibition because, when the early portion of the delay CS was interrupted shortly by an empty interval, the difference in the time of maximum responding between the lesioned and control subjects was eliminated. Thus, although the involvement of the DHPC in appetitive trace conditioning was not found when a gross measure of conditioning was employed, it was revealed when the temporal distribution of conditioned responding was examined on a moment-by-moment basis as in eyeblink trace conditioning studies.     

Pavlovian aversive and appetitive odor conditioning in humans: subjective, peripheral, and electrocortical changes

The effects of presynaptic guanosine-5'-O-(3-thio)triphosphate (GTPγS) on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in cultured hippocampal neurons using whole-cell recordings. Inclusion of GTPγS (0.5–1 mM) in the presynaptic electrode reduced both the amplitude and paired-pulse depression of IPSCs, indicating that the probability of GABA-release had been reduced. Presynaptic GTPγS increased the depression of IPSCs by the GABA_B-receptor-agonist baclofen (10 μM), and the effect of baclofen was poorly reversible after washing. Stimulation of the GABAergic neuron at 80 Hz for 1 s was accompanied by tetanic depression of the IPSCs by 52±6% and was followed by post-tetanic potentiation (PTP), reaching a peak value of 71±21% and lasting about 100 s. IPSCs evoked after tetanic stimulation were depressed and PTP was absent when tetanic stimulation was applied within 3 min after starting injection of GTPγS into the presynaptic neuron. At longer times, basal release underlying a single IPSC was depressed. This affected the ratios recorded in response to tetanic stimulations such that tetanic depression was abolished, while PTP increased to 117±34%. In conclusion, GTPγS reduces the probability of GABA-release in both a use- and time-dependent manner, most likely through an inhibitory action on presynaptic Ca²⁺-influx through voltage-gated Ca²⁺ channels or an interaction with small GTP-binding proteins in the nerve terminals. Electronic Publication     

Procedural memory system supports single cue trace eyeblink conditioning in medial temporal lobe amnesia

A number of studies investigating trace eyeblink conditioning have found impaired, but not eliminated, acquisition of conditioned responses (CRs) in both animals and humans with hippocampal removal or damage. The underlying mechanism of this residual learning is unclear. The present study investigated whether the impaired level of learning is the product of residual hippocampal function or whether it is mediated by another memory system that has been shown to function normally in delay eyeblink conditioning. Performance of bilateral medial temporal lobe amnesic patients who had a prior history of participating in eyeblink conditioning studies was compared to a control group with a similar training history and to an untrained control group in a series of single cue trace conditioning tasks with 500 ms, 250 ms, and 0 ms trace intervals. Overall, patients acquired CRs to a level similar to the untrained controls, but were significantly impaired compared to the trained controls. The pattern of acquisition suggests that amnesic patients may be relying on the expression of previously acquired, likely cerebellar based, procedural memory representations in trace conditioning.     

Dorsal hippocampal involvement in conditioned-response timing and maintenance of temporal information in the absence of the CS

Involvement of the dorsal hippocampus (DHPC) in conditioned-response timing and maintaining temporal information across time gaps was examined in an appetitive Pavlovian conditioning task, in which rats with sham and DHPC lesions were first conditioned to a 15-s visual cue. After acquisition, the subjects received a series of non-reinforced test trials, on which the visual cue was extended (45 s) and gaps of different duration, 0.5, 2.5, and 7.5 s, interrupted the early portion of the cue. Dorsal hippocampal-lesioned subjects underestimated the target duration of 15 s and showed broader response distributions than the control subjects on the no-gap trials in the first few blocks of test, but the accuracy and precision of their timing reached the level of that of the control subjects by the last block. On the gap trials, the DHPC-lesioned subjects showed greater rightward shifts in response distributions than the control subjects. We discussed these lesion effects in terms of temporal versus non-temporal processing (response inhibition, generalisation decrement, and inhibitory conditioning).     

Spontaneous recovery but not reinstatement of the extinguished conditioned eyeblink response in the rat

Reinstatement--the return of an extinguished conditioned response (CR) after reexposure to the unconditioned stimulus (US)--and spontaneous recovery--the return of an extinguished CR with the passage of time--are 2 of 4 well-established phenomena that demonstrate that extinction does not erase the conditioned stimulus (CS)-US association. However, reinstatement of extinguished eyeblink CRs has never been demonstrated, and spontaneous recovery of extinguished eyeblink CRs has not been systematically demonstrated in rodent eyeblink conditioning. In Experiment 1, US reexposure was administered 24 hr prior to a reinstatement test. In Experiment 2, US reexposure was administered 5 min prior to a reinstatement test. In Experiment 3, a long, discrete cue (a houselight), present in all phases of training and testing, served as a context within which each trial occurred to maximize context processing, which in other preparations has been shown to be required for reinstatement. In Experiment 4, an additional group was included that received footshock exposure, rather than US reexposure, between extinction and test, and contextual freezing was measured prior to test. Spontaneous recovery was robust in Experiments 3 and 4. In Experiment 4, context freezing was strong in a group given footshock exposure but not in a group given eye shock US reexposure. There was no reinstatement observed in any experiment. With stimulus conditions that produce eyeblink conditioning and research designs that produce reinstatement in other forms of classical conditioning, we observed spontaneous recovery but not reinstatement of extinguished eyeblink CRs. This suggests that reinstatement, but not spontaneous recovery, is a preparation- or substrate-dependent phenomenon.     

Generalization of appetitive conditioned responses

A stimulus (conditioned stimulus, CS) associated with an appetitive unconditioned stimulus (US) acquires positive properties and elicits appetitive conditioned responses (CR). Such associative learning has been examined extensively in animals with food as the US, and results are used to explain psychopathologies (e.g., substance‐related disorders or obesity). Human studies on appetitive conditioning exist, too, but we still know little about generalization processes. Understanding these processes may explain why stimuli not associated with a drug, for instance, can elicit craving. Forty‐seven hungry participants underwent an appetitive conditioning protocol during which one of two circles with different diameters (CS+) became associated with an appetitive US (chocolate or salty pretzel, according to participants’ preference) but never the other circle (CS?). During generalization, US were delivered twice and the two CS were presented again plus four circles (generalization stimuli, GS) with gradually increasing diameters from CS? to CS+. We found successful appetitive conditioning as reflected in appetitive subjective ratings (positive valence, higher contingency) and physiological responses (startle attenuation and larger skin conductance responses) to CS+ versus CS?, and, importantly, both measures confirmed generalization as indicated by generalization gradients. Small changes in CS‐US contingency during generalization may have weakened generalization processes on the physiological level. Considering that appetitive conditioned responses can be generalized to non‐US‐associated stimuli, a next important step would be to investigate risk factors that mediate overgeneralization.     

Enhanced sensitization to animal,interpersonal, and intergroup fear‐relevant stimuli (but no evidence for selective one‐trial fear learning)

Selective sensitization has been proposed as an alternative explanation for enhanced responding to animal fear‐relevant stimuli—snakes and spiders—during extinction of Pavlovian fear conditioning. The current study sought to replicate the phenomenon using a shock workup procedure as the sensitizing manipulation and to extend it to interpersonal and intergroup fear‐relevant stimuli—angry faces and other‐race faces. Assessment of selective sensitization was followed by a one‐trial fear learning procedure. Selective sensitization, larger electrodermal responses to fear‐relevant than to control stimuli after sensitization, or a larger increase in electrodermal responding to fear‐relevant than to control stimuli after sensitization was observed across stimulus domains. However, the one‐trial fear learning procedure failed to provide evidence for enhanced fear conditioning to fear‐relevant stimuli. One‐trial fear learning was either absent or present for fear‐relevant and nonfear‐relevant stimuli. The current study confirms that electrodermal responses to fear‐relevant stimuli across stimulus domains are subject to selective sensitization.     

Cerebellum lesion impairs eyeblink-like classical conditioning in goldfish

The cerebellum of mammals is an essential component of the neural circuitry underlying classical conditioning of eyeblink and other discrete responses. Although the neuroanatomical organization of the cerebellum is notably well conserved in vertebrates, little is actually known about the cerebellar learning functions in nonmammal vertebrate groups. In this work we studied whether the cerebellum of teleost fish plays a critical role in the classical conditioning of a motor response. In Experiment 1, we classically conditioned goldfish in a procedure analogous to the eyeblink conditioning paradigm commonly used in mammals. Goldfish were able to learn to express an eyeblink-like conditioned response to a predictive light (conditioned stimulus) that was paired with a mild electric shock (unconditioned stimulus). The application of unpaired and extinction control procedures demonstrated that also in teleosts the learning of this motor response depends on associative rules. In Experiment 2 we studied whether classical conditioning of this response is critically dependent on the cerebellum and independent of telencephalic structures as occurs in mammals. Cerebellum lesion prevented the acquisition of the eyeblink-like conditioned response whereas telencephalon ablation did not impair the learning of this response. No deficit was observed following lesions in the performance of the unconditioned response or in the percentage of spontaneous responses. These results suggest that cerebellum ablation in goldfish affects a critical component of the circuitry necessary for the acquisition of the conditioned response but does not interfere with the ability of the animal to perform the response itself. The striking similarity in the role of cerebellum in classical conditioning of a motor response between teleost fish and mammals suggests that this learning function of the cerebellum could be a primitive feature of the vertebrate brain that has been conserved through evolution.     

Learning to fear suffocation: a new paradigm for interoceptive fear conditioning

The present study aimed to establish a new interoceptive fear conditioning paradigm. The conditioned stimulus (CS) was a flow resistor that slightly obstructs breathing; the unconditional stimulus (US) was a breathing occlusion. The paired group (N = 21) received 6 acquisition trials with paired CS–US presentations. The unpaired group (N = 19) received 6 trials of unpaired CS–US presentations. In the extinction phase, both groups were administered 6 CS‐only trials. Measurements included startle eyeblink response, electrodermal responses, and self‐reported US expectancy. In the paired group, startle blink responses were larger during CS compared to intertrial interval during acquisition and extinction. Electrodermal and US expectancies were larger for the paired than for the unpaired group during acquisition, but not during extinction. The present paradigm successfully established interoceptive fear conditioning with panic‐relevant stimuli.