Selective expression of electrical correlates of differential appetitive classical conditioning in a feeding network

Electrical correlates of differential appetitive classical conditioning were recorded in the neural network that underlies feeding in the snail Lymnaea stagnalis. In spaced training (15 trials over 3 days), the lips and the tentacle were used as CS+ (reinforced conditioned stimulus) or CS- (nonreinforced conditioned stimulus) sites for behavioral tactile conditioning. In one group of experimental animals, touch to the lips (the CS+ site) was followed by sucrose (the unconditioned stimulus, US), but touch to the tentacle (the CS- site) was not reinforced. In a second experimental group the CS+/CS- sites were reversed. Semi-intact lip-tentacle-CNS preparations were made from both experimental groups and a naive control group. Intracellular recordings were made from the B3 motor neuron of the feeding network, which allowed the monitoring of activity in the feeding central pattern generator (CPG) interneurons as well as early synaptic inputs evoked by the touch stimulus. Following successful behavioral conditioning, the touch stimulus evoked CPG-driven fictive feeding activity at the CS+ but not the CS- sites in both experimental groups. Naive snails/preparations showed no touch responses. A weak asymmetrical stimulus generalization of conditioned feeding was not retained at the electrophysiological level. An early excitatory postsynaptic potential (EPSP) response to touch was only enhanced following conditioning in the Lip CS+/tentacle CS- group but not in the Tentacle CS+/lip CS- group. The results show that the main features of differential appetitive classical conditioning can be recorded at the electrophysiological level, but some characteristics of the conditioned response are selectively expressed in the reduced preparation.     

Reduced amygdala responsivity during conditioning to trauma‐related stimuli in posttraumatic stress disorder

Exaggerated conditioned fear responses and impaired extinction along with amygdala overactivation have been observed in posttraumatic stress disorder (PTSD). These fear responses might be triggered by cues related to the trauma through higher‐order conditioning, where reminders of the trauma may serve as unconditioned stimuli (US) and could maintain the fear response. We compared arousal, valence, and US expectancy ratings and BOLD brain responses using fMRI in 14 traumatized persons with PTSD and 14 without PTSD (NPTSD) and 13 matched healthy controls (HC) in a differential aversive conditioning paradigm. The US were trauma‐specific pictures for the PTSD and NPTSD group and equally aversive and arousing for the HC; the conditioned stimuli (CS) were graphic displays. During conditioning, the PTSD patients compared to the NPTSD and HC indicated higher arousal to the conditioned stimulus that was paired with the trauma picture (CS+) compared to the unpaired (CS?), increased dissociation during acquisition and extinction, and failure to extinguish the CS/US‐association compared to NPTSD. During early and late acquisition, the PTSD patients showed a significantly lower amygdala activation to CS+ versus CS? and a negative interaction between activation in the amygdala and dorsolateral prefrontal cortex (PFC), while NPTSD and HC displayed a negative interaction between amygdala and medial PFC. These findings suggest maladaptive anticipatory coping with trauma‐related stimuli in patients with PTSD, indicated by enhanced conditioning, with related abnormal amygdala reactivity and connectivity, and delayed extinction.     

Conditioned and extinguished fear modulate functional corticocardiac coupling in humans

Although the conditioned cardiac fear response is an important index of psychophysiological fear processing, underlying neural mechanisms remain unclear. N = 22 participants underwent differential fear conditioning and extinction with face pictures as conditioned stimuli (CS) and loud noise bursts as aversive unconditioned stimulus (US) on Day 1 and a recall test 1 day later. We assessed ERPs, evoked heart period (HP), and time‐lagged within‐subject correlations of single‐trial EEG amplitude and HP as index for corticocardiac coupling in response to the CS. Fear‐conditioned stimuli (CS+) triggered cardiac deceleration during fear acquisition and recall. Meanwhile, only during Day 1 acquisition, CS+ evoked larger late positivities in the ERP than CS?. Most importantly, during Day 2 recall, stimulus‐evoked single‐trial EEG responses in the time window between 250 and 500 ms predicted the magnitude of cardiac fear responses 2 to 5 s later. This marker of corticocardiac coupling selectively emerged in response to not previously extinguished CS+ but was absent in response to CS? or previously extinguished CS+. The present results provide first evidence that fear conditioning and extinction modulate functional corticocardiac coupling in humans. Underlying mechanisms may involve subcortical structures enhancing corticocardiac transmission to facilitate processing of consolidated conditioned fear.     

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.     

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.     

Multiple functions of context during conditioning: a developmental analysis

Contextual stimuli may influence conditioned behavior in at least two ways (e.g. Bouton & Bolles, 1985). By becoming associated with the unconditioned stimulus (US), context cues can acquire excitatory strength that facilitates responding to a phasic conditioned stimulus (CS). The context also can function to clarify the meaning of an ambiguous CS. Data obtained with an appetitive Pavlovian conditioning paradigm suggest that the processes mediating these two influences of context are dissociated during development. Evidence of context-US associations was observed in rats that began training on Postnatal Day 17, but no evidence for a disambiguation function was found until pups were 20- to 23-days-old. Evidence for a context-US association was obtained by demonstrating that US alone presentations in the training context restored conditioned responding to an extinguished CS. Evidence for a disambiguation function was obtained by demonstrating that a context shift, concurrent with extinction of responding to a phasic CS, preserved responding to the CS when the subjects were subsequently tested in the training context. These findings were discussed in relation to (a) the development of the rat's ability to use relational representations, and (b) Nadel and Zola-Morgan's (1984) hypothesis linking hippocampal maturation to the role of context during development.     

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     

Fear potentiated startle at short intervals following conditioned stimulus onset during delay but not trace conditioning

The latency of conditioned fear after delay and trace conditioning was investigated. Some argue that delay conditioning is not dependent on awareness. In contrast, trace conditioning, where there is a gap between the conditioned stimulus (CS) and the unconditioned stimulus (US), is assumed to be dependent on awareness. In the present study, a tone CS signaled a noise US presented 1000 ms after CS onset in the delay conditioning group. In the trace conditioning group, a 200-ms tone CS was followed by an 800-ms gap prior to US presentation. Fear-potentiated startle should be seen at shorter intervals after delay conditioning compared to trace conditioning. Analyses showed increased startle at 30, 50, 100, and 150 ms after CS onset following delay conditioning compared to trace conditioning. This implies that fear-relevant stimuli elicit physiological reactions before extended processing of the stimuli occur, following delay, but not trace conditioning.     

Awareness of the CS-UCS contingency and classical conditioning of skin conductance responses with olfactory CSs

The possibility of demonstrating acquisition of classically conditioned responses without awareness of the conditioned stimulus-unconditioned stimulus (CS-UCS) contingency using olfactory stimuli with 58 college student subjects was tested. A classical discrimination delay conditioning paradigm was employed, with electric shock as the UCS and two pleasant odors (perfumes) as the conditioned stimuli (CS+ and CS-). Trial-by-trial measures of skin conductance conditioned responses served as dependent variables. A masking task in the form of an olfactory memory task was employed for the purpose of delaying the onset of awareness of the conditioning contingency. Awareness of the conditioning contingency was assessed by a concurrent and a post hoc measure, and subjects who satisfied both criteria were considered aware of the CS-UCS contingency. Conditioning was observed only in the aware subjects, and only after the onset of awareness of the CS+-UCS contingency. Respiratory activity, measured as a check against possible artifacts, had no effect on the SCR measures. It was concluded that the awareness of the CS-UCS contingency is necessary for acquisition of discriminative conditioned responses in humans, regardless of the sensory modality in which CSs are presented. Sex differences in skin conductance measures and performance on the olfactory memory task were observed.     

Appetitive-aversive interactions in Pavlovian fear conditioning

The existence of value coding and salience coding neurons in the mammalian brain, including in habenula and ventral tegmental area, has sparked considerable interest in the interactions that occur between Pavlovian appetitive and aversive conditioning. Here we studied these appetitive-aversive interactions at the behavioral level by assessing the learning that occurs when a Pavlovian appetitive conditioned stimulus (conditional stimulus, CS) serves as a CS for shock in Pavlovian fear conditioning. A Pavlovian appetitive CS was retarded in the rate at which it could be transformed into a fear CS (counterconditioning), but the presence of the appetitive CS augmented fear learning to a concurrently presented neutral CS (superconditioning). Retardation of fear learning was not alleviated by manipulations designed to restore the associability of the appetitive CS before fear conditioning but was alleviated by manipulations designed to increase the aversive quality of the shock unconditioned stimulus (US). These findings are consistent with opponent interactions between the appetitive and aversive motivational systems and provide a behavioral approach for assessing the neural correlates of these appetitive-aversive interactions.     

Sleep deprivation increases threat beliefs in human fear conditioning

Sleep disturbances and anxiety disorders exhibit high comorbidity levels, but it remains unclear whether sleep problems are causes or consequences of increased anxiety. To experimentally probe the aetiological role of sleep disturbances in anxiety, we investigated in healthy participants how total sleep deprivation influences fear expression in a conditioning paradigm. In a fear conditioning procedure, one face stimulus (conditioned stimulus [CS+]) was paired with electric shock, whereas another face stimulus was not (unpaired stimulus [CS?]). Fear expression was tested the next morning using the two face stimuli from the training phase and a generalization stimulus (i.e. a morph between the CS+ and CS? stimuli). Between fear conditioning and test, participants were either kept awake in the laboratory for 12 hr (n = 20) or had a night of sleep at home (n = 20). Irrespective of stimulus type, subjective threat expectancies, but not skin conductance responses, were enhanced after sleep deprivation, relative to regular sleep. These results suggest that sleep disturbances may play a role in anxiety disorders by increasing perceived threat.     

Fear acquisition requires awareness in trace but not delay conditioning

The present study explored fear acquisition in differential delay versus trace conditioning in regard to the potential role of the acquired contingency awareness. One of two neutral pictures (CS+) either coterminated with (delay group; n=32) or was followed by the aversive unconditioned stimulus (UCS) after CS offset (trace group; n=32), while startle blink and skin conductance responses (SCR) were measured. As expected, the acquisition of conditioned startle potentiation in delay conditioning was independent of contingency awareness. In contrast, fear-potentiated startle in trace conditioning was only observed for those participants who were aware of the CS-UCS contingencies. SCR conditioning was generally only obtained for aware participants. The present results suggest a more implicit learning process in delay fear conditioning, whereas the explicit acquisition of contingency awareness might be a prerequisite for trace fear conditioning.     

Neural, electrodermal and behavioral response patterns in contingency aware and unaware subjects during a picture-picture conditioning paradigm

One way of investigating affective learning is the use of aversive pictures as unconditioned stimuli (UCS) in conditioning paradigms. In the last decades, there has been a heated debate on the influence of contingency awareness on conditioned responses (CRs). Only a few studies found CRs in contingency unaware subjects whereas other studies only reported conditioned reactions in contingency aware participants. However, as a shortcoming, most studies employing picture-picture paradigms only investigated one response level (e.g. changes in subjective ratings). Further, changes in brain activity have so far been neglected in this field of research. The aim of the present study was to investigate different response levels with respect to contingency awareness: brain activity measured by functional magnetic resonance imaging (fMRI), skin conductance responses (SCRs) and valence ratings. A neutral geometric shape (conditioned stimulus, CS+) was followed by aversive pictures, whereas another shape (CS-) preceded neutral pictures. Unaware participants showed CRs in brain activity (e.g. the insula). Generally more activity was observed in the fear network (e.g. the amygdala, the lateral orbitofrontal cortex) in aware participants and in the nucleus accumbens (NAcc). Investigation of SCRs and valence ratings revealed that only aware participants showed conditioned reactions. Our results point toward dissociations between response levels (e.g. brain activity) not affected by contingency awareness and more cognitive response levels (e.g. subjective ratings and SCRs) which are affected by contingency awareness. As a unique finding in human aversive conditioning, we discuss the role of the nucleus accumbens as well as practical implications for affective learning models.     

Classical conditioning in oddball paradigm: A comparison between aversive and name conditioning

The nature of cortical plasticity in learning is one of the most intriguing questions of modern cognitive neuroscience. Classical conditioning (as a typical case of associative learning) and electroencephalography together provide a good framework for expanding our knowledge about fast learning‐related cortical changes. In our experiment, we employed a novel paradigm in which classical conditioning was combined with passive oddball. Nineteen subjects participated in the first experiment (aversive conditioning with painful shock as unconditioned stimulus (US) and neutral tones as conditioned stimulus (CS)), and 22 subjects in the second experiment (with a subject’s own name as US). We used event‐related potentials (ERPs) and time‐frequency analyses to explore the CS‐US interaction. We found a learning‐induced increment of P3a in the first experiment and the late positive potential (LPP) in both experiments. These effects may be related to increased attentional and emotional significance of conditioned stimuli. We showed that the LPP and P3a effects, earlier found only in visual paradigms, generalize to the auditory sensory system. We also observed suppression of the low beta activity to CS+ in aversive conditioning over the hemisphere contralateral to expected electrical shocks, presumably indicating preparation of the somatosensory system to the expected nociceptive US.     

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.     

The development of fear learning and generalization in 8-13 year-olds

The current study examined developmental changes in fear learning and generalization in 40 healthy 8–13 year‐olds using an aversive conditioning paradigm adapted from Lau et al. [Lau et al. [2008] Journal of the American Academy of Child and Adolescent Psychiatry 47:94–102]. In this task, the conditioned stimuli (CS+/CS?) are two neutral female faces, and the unconditioned stimulus is a fearful, screaming face. The second phase of the study also included a generalization stimulus (GS): a 50% blend of the CS± faces. The eye‐blink startle reflex was utilized to measure defensive responding. Patterns of fear learning and generalization were qualified by child age. Older children demonstrated greater fear learning (i.e., larger startle during CS+ than CS?) than younger children. In addition, older children exhibited the typical pattern of generalization observed in adults, whereas younger children did not. Finally, fear learning also related to contingency awareness; only children who correctly identified the CS+ demonstrated fear‐potentiated startle to the CS+. Clinical implications and future directions are discussed. © 2011 Wiley Periodicals,Inc. Dev Psychobiol 54: 675–684, 2012.     

Effects of stimulus modality and task condition on blink startle modification and on electrodermal responses

Participants in Experiments 1 and 2 performed a discrimination and counting task to assess the effect of lead stimulus modality on attentional modification of the acoustic startle reflex. Modality of the discrimination stimuli was changed across subjects. Electrodermal responses were larger during task-relevant stimuli than during task-irrelevant stimuli in all conditions. Larger blink magnitude facilitation was found during auditory and visual task-relevant stimuli, but not for tactile stimuli. Experiment 3 used acoustic, visual, and tactile conditioned stimuli (CSs) in differential conditioning with an aversive unconditioned stimulus (US). Startle magnitude facilitation and electrodermal responses were larger during a CS that preceded the US than during a CS that was presented alone regardless of lead stimulus modality. Although not unequivocal, the present data pose problems for attentional accounts of blink modification that emphasize the importance of lead stimulus modality.     

Single neurons in the medial prefrontal cortex of the rat exhibit tonic and phasic coding during trace fear conditioning

Trace fear conditioning is a learning task that requires the association of an auditory conditioned stimulus (CS) and a shock unconditioned stimulus (US) that are separated by a 20-s trace interval. Single neuron activity was recorded from the prelimbic and infralimbic areas of the medial prefrontal cortex in rats during trace fear conditioning or nonassociative unpaired training. Prelimbic neurons showed learning-related increases in activity to the CS and US, whereas infralimbic neurons showed learning-related decreases in activity to these stimuli. A subset of prelimbic neurons exhibited sustained increases in activity during the trace interval. These sustained prelimbic responses may provide a bridging code that allows for overlapping representations of CS and US information within the trace fear conditioning circuit.     

Awareness is necessary for differential trace and delay eyeblink conditioning in humans

Squire et al. have proposed that trace and delay eyeblink conditioning procedures engage separate learning systems: a declarative hippocampal/cortical system associated with conscious contingency awareness, and a reflexive sub-cortical system independent of awareness, respectively ( [Clark and Squire, 1998] and [Smith et al., 2005]). The only difference between these two procedures is that the conditioned stimulus (CS) and the unconditioned stimulus (US) overlap in delay conditioning, whereas there is a brief interval (e.g., 1 s) between them in trace conditioning. In two experiments using the same procedure as Clark and Squire's group, we observed differential conditioning only in participants who showed contingency awareness in a post-experimental questionnaire, with both trace and delay procedures. We interpret these results to suggest that, although there may be multiple brain regions involved in learning, these regions are organized as a coordinated system rather than as separate, independent systems.     

Development of learned flavor preferences

Rats, like humans, are born with only a few innate flavor preferences and aversions. Preferences retain great plasticity throughout the lifespan because they are sensitive to modification by experience. From an early age, rats can rapidly learn to prefer or avoid a flavor (conditioned stimulus, CS) that is associated with a positive or negative unconditioned stimulus (US). The US may be the mother's milk, social or thermotactile stimulation, or other food-related stimuli. Flavor-flavor learning occurs when the CS flavor is mixed with a naturally preferred (e.g., sweet) or avoided (e.g., bitter) US flavor. Flavor preferences and aversions are also produced by USs that have postoral positive (e.g., nutritious) or negative (e.g., toxic) actions. These types of learning appear to involve different behavioral and neural mechanisms as indicated by differences in conditioned responses, effective temporal parameters, resistance to extinction, and neurochemical mechanisms. New evidence indicates that flavor-nutrient preference learning can occur before weaning and influence food selection after weaning. Flavor conditioning not only affects food choice, but can also significantly increase food acceptance, that is, total consumption. Thus, from an early age, learning processes shape the feeding behavior of animals. While primarily serving an adaptive function, learning may play a role in biasing individuals towards excessive intake and weight gain.