Salivary conditioning with antennal gustatory unconditioned stimulus in an insect

Classical conditioning of olfactory conditioning stimulus (CS) with gustatory unconditioned stimulus (US) in insects has been used as a pertinent model for elucidation of neural mechanisms underlying learning and memory. However, a conditioning system in which stable intracellular recordings from brain neurons are feasibly obtained while monitoring the conditioning effect has remained to be established. Recently, we found classical conditioning of salivation in cockroaches Periplaneta americana, in which an odor was associated with sucrose solution applied to the mouth, and this conditioning could be monitored by activities of salivary neurons. Application of gustatory US to the mouth, however, leads to feeding movement accompanying a movement of the brain that prevents stable recordings from brain neurons. Here we investigated whether a gustatory stimulus presented to an antenna could serve as an effective US for producing salivary conditioning. Presentation of sucrose or sodium chloride solution to an antenna induced salivation and also increased activities of salivary neurons. A single pairing trial of an odor with antennal presentation of sucrose or sodium chloride solution produced conditioning of salivation or of activities of salivary neurons. Five pairing trials led to a conditioning effect that lasted for one day. Water or tactile stimulus presented to an antenna was not effective for producing conditioning. The results demonstrate that gustatory US presented to an antenna is as effective as that presented to the mouth for producing salivary conditioning. This conditioning system provides a useful model for studying the neural basis of learning at the level of singly identifiable neurons.     

An immediate-shock freezing deficit with discrete cues: a possible role for unconditioned stimulus processing mechanisms.

Five experiments with C57BL/6 mice (Mus musculus) investigated whether failures in shock processing might contribute to deficits in freezing that occur after an animal receives a shock immediately on exposure to a conditioning context. Experiment 1 found that more contextual freezing resulted from delayed shocks than from immediate shocks across 4 shock intensities. Experiment 2 extended the immediate-shock freezing deficit to discrete stimuli. Experiment 3 found that preexposure to the to-be-conditioned cue did not facilitate immediate cued conditioning. Experiment 4 found that context preexposure enhanced context-evoked fear after an immediate shock. Experiment 5 found that context preexposure also enhanced immediate cued conditioning. These findings are problematic for current theories of the immediate-shock freezing deficit that focus exclusively on processing of the conditioned stimulus, and they suggest that failures in shock processing may contribute to the deficit.     

Effect of changing the unconditioned stimulus on appetitive blocking

In four experiments we examined the effects of changing the unconditioned stimulus (US) on appetitive blocking. In Experiments 1A, 1B, and 2 we established that substituting food for water, or water for food, in the compound stage did not attenuate blocking relative to groups that received the same US throughout conditioning. Experiment 3 showed that satiation with the US used prior to compound conditioning with a different US does not affect blocking. Experiment 4 revealed that changing the location of US delivery, as well as the quality of the US, also leaves blocking unaffected. It is suggested that these results demonstrate that blocking occurs, provided that there is no change in the affective properties of the US.     

Conditioned response-contingent delays of the unconditioned stimulus in human aversive conditioning.

Four groups of subjects were given either 0. 100, 500, or 1,000 msec delays of the unconditioned stimulus (UCS) contingent upon the occurrence of a conditioned response (CR) and were given a UCS 515 msec after conditioned stimulus (CS) onset when a CR did not occur. A fifth group received standard classical conditioning trials with an interstimulus interval of 515 msec. Overall performance decreased as CR-contingent UCS delay increased, with the classical conditioning group approximating the performance of the group receiving the 100-msec delay. The data were analyzed with the two-phase model of conditioning and the following results were obtained: The duration of Phase 1 of the model increased with contingent delay; operator limits associated with CR trials or with combined CR-CR (CR absent) trials decreased as a function of delay; and operator limits associated exclusively with CR trials were unaffected by the delay. Subjects receiving a contingent delay of 0 msec gave the shortest latency responses and exhibited reliable latency decreases across trials, suggesting an attempt to "beat" the UCS. The results were interpreted as contrary to what would be expected from low-of-effect theories which postulate that reinforcement results from a CR-UCS interaction, although they could be subsumed under a drive or an associative strength theory in which the aversive, or CR-supportive, strength of the UCS is assumed to be negatively correlated with contingent UCS delay.     

Effect of preconditioning unconditioned stimulus experience on learned taste aversions.

One taste-aversion study using male Long-Evans rats in which ethanol was the unconditioned stimulus (UCS) and six studies in which lithium chloride (LiCl) was the UCS demonstrate that (a) exposure to the UCS prior to conditioning retards subsequent acquisition of learned taste aversions; (b) a single preconditioning UCS exposure is sufficient to attenuate conditioning; (c) the preconditioning UCS exposure must occur within a limited period prior to conditioning to attenuate learning; (d) repeated conditioning trials will override the effect of prior exposure to the UCS; (e) tolerance to the UCS is not a necessary condition for the attenuation effect to occur; (f) pairing the preconditioning UCS with a novel flavor other than the CS does not remove the preexposure effect, although it may reduce its magnitude; and (g) the degree of disruption is a positive function of preconditioning UCS dosage and an inverse function of conditioning UCS dosage.     

Pentylenetetrazole as an unconditioned stimulus for olfactory and contextual fear conditioning in rats

The association of five footshocks with a neutral odor is able to establish an olfactory fear conditioning in rats. The present study sought to investigate whether the systemic administration of pentylenetetrazole (PTZ; 3.75–15 mg/kg) would turn the coffee odor in a conditioned stimulus in the fear conditioning paradigm. The results showed that rats started to display risk assessment and avoidance after PTZ (15 mg/kg)–coffee odor pairing. When three mild footshocks (0.4 mA for 2 s) were delivered during this pairing, the conditioned response exhibited was greater than before. In both cases, however, pretreatment with the benzodiazepine midazolam (MDZ. 0.5 mg/kg i.p.) fully counteracted the expression of these defensive behaviors. Moreover, after being paired with 15 mg/kg of PTZ alone or combined with footshocks, the coffee odor was able to promote a new fear conditioning related to the context where it was re-exposed. The present findings point out the usefulness of PTZ as an unconditioned stimulus to promote fear conditioning to olfactory and contextual cues in rats.     

Effect of unconditioned stimulus magnitude on the emergence of conditioned responding

Although unconditioned stimulus (US) magnitude influences conditioning, no experiment has addressed whether it influences a decision point at which the organism first responds (Gallistel & Gibbon, 2000). Two appetitive conditioning experiments with rats confirmed that the rate of food cup entries and proportion of head jerking were related to the number of pellets (US) provided after the conditioned stimulus. In addition, the onset of responding measured by the number of reinforcers to a criterion or by a quantitatively identified change point also reflected US magnitude. Two aversive conditioning experiments also found that the amount and onset of freezing were related to footshock intensity. All experiments identified a trial at which responding increased abruptly in individual subjects. However, the point where the increase occurred was earlier with larger USs.     

Information about the model's unconditioned stimulus and response in vicarious classical conditioning.

Four groups with 16 observers each participated in a differential, vicarious conditioning experiment with skin conductance responses as the dependent variable. The information available to the observer about the model's unconditioned stimulus and response was varied in a 2 X 2 factorial design. Results clearly showed that information about the model's unconditioned stimulus (a high or low dB level) was not necessary for vicarious instigation, but that information about the unconditioned response (a high or low emotional aversiveness) was necessary. Data for conditioning of responses showed almost identical patterns to those for vicarious instigation. To explain the results, a distinction between factors necessary for the development and elicitation of vicariously instigated responses was introduced, and the effectiveness of information about the model's response on the elicitation of vicariously instigated responses was considered in terms of an expansion of Bandura's social learning theory.     

Modulation of unconditioned defense reflexes by a putative emotive Pavlovian conditioned stimulus

Four experiments showed differential modulation of defensive unconditioned responses (URs) in rabbits by contextual stimuli that Brandon and Wagner (1991) have shown similarly to modulate conditioned eyeblink responses. Two 30-s auditory cues, A and B, were differentially paired with shock. Tests were presentations of a response-eliciting probe stimulus within A, B, or a comparable blank interval, Experiments 1 and 2 demonstrated that A and B differentially facilitated eyeblink URs, and Experiments 3 and 4 showed that A and B similarly differentially facilitated startle responses elicited by airpuffs to the ear. These data are consistent with a characterization of Pavlovian conditioning that distinguishes between emotive and sensory conditioning and assumes that conditioned emotional responses similarly modulate specific conditioned and unconditioned defensive reflexes (Konorski, 1967; Wagner & Brandon, 1989).     

Lithium-mediated disruptions of latent inhibition: Overshadowing by the unconditioned stimulus in flavor conditioning

In a series of experiments, the ability of a single lithium preexposure to disrupt CS effectiveness was assessed using a latent inhibition procedure. Lithium preexposure administered proximal (90 min) to a saccharin familiarization trial reduced latent inhibition whereas a similar administration more distal (360 min) to flavor familiarization failed to do so. Additional experiments demonstrated that this socalled “US overshadowing” effect was not attributable to sensitization (Experiment 2), excitatory backward conditioning (Experiment 3), or state dependency (Experiment 4). The implications of US overshadowing for proximal US-preexposure effects are discussed.     

Conditioned taste aversion is enhanced when the unconditioned stimulus is presented in a different context

Using a conditioned taste aversion procedure with rats as the subjects, two experiments examined the effect of presenting a conditioned stimulus (CS saccharin solution) in one context followed by an unconditioned stimulus (US LiCl) in a different context. Experiment 1 showed that animals which received the above-mentioned procedure (Group D) showed a more marked conditioned aversion to the CS than animals which were given both the CS and the US in the same context (Group S). Experiment 2 found that in both Group D and Group S, aversion to the CS increased when the subjects were exposed to the conditioned context after the conditioning. These findings supported the argument that the strength of the CS-US association acquired during conditioning is compared with that of the context-US to determine the magnitude of aversion revealed to the CS.     

The amygdala is not necessary for unconditioned stimulus inflation after Pavlovian fear conditioning in rats

The basolateral complex (BLA) and central nucleus (CEA) of the amygdala play critical roles in associative learning, including Pavlovian conditioning. However, the precise role for these structures in Pavlovian conditioning is not clear. Recent work in appetitive conditioning paradigms suggests that the amygdala, particularly the BLA, has an important role in representing the value of the unconditioned stimulus (US). It is not known whether the amygdala performs such a function in aversive paradigms, such as Pavlovian fear conditioning in rats. To address this issue, Experiments 1 and 2 used temporary pharmacological inactivation of the amygdala prior to a US inflation procedure to assess its role in revaluing shock USs after either overtraining (Experiment 1) or limited training (Experiment 2), respectively. Inactivation of the BLA or CEA during the inflation session did not affect subsequent increases in conditioned freezing observed to either the tone conditioned stimulus (CS) or the conditioning context in either experiment. In Experiment 3, NBQX infusions into the BLA impaired the acquisition of auditory fear conditioning with an inflation-magnitude US, indicating that the amygdala is required for associative learning with intense USs. Together, these results suggest that the amygdala is not required for revaluing an aversive US despite being required for the acquisition of fear to that US.Pavlovian fear conditioning in rats is a behavioral model used to investigate the neurobiology underlying the development and maintenance of fear learning and memory (Grillon et al. 1996; LeDoux 1998, 2000; Bouton et al. 2001; Maren 2001b, 2005; Kim and Jung 2006). In this model, an innocuous conditioned stimulus (CS), such as a tone, is paired with an aversive unconditioned stimulus (US), such as a footshock. After one or more pairings, the rat learns that the CS predicts the US. As a consequence, CS presentations alone elicit a conditioned fear response (CR), which includes increases in heart rate, arterial blood pressure, hypoalgesia, potentiated acoustic startle, stress hormone release, and freezing (somatomotor immobility).The amygdala has been identified as one of the major regions in which fear memories are encoded and stored. Within the amygdala, the basolateral complex of the amygdala (BLA; consisting of the lateral, basolateral, and basomedial nuclei) and the central nucleus of the amygdala (CEA) receive convergent CS and US information and are involved in the acquisition of fear memories (LeDoux 1998, 2000; Fendt and Fanselow 1999; Davis and Whalen 2001; Maren 2001b; Schafe et al. 2001; Fanselow and Gale 2003; Wilensky et al. 2006; Zimmerman et al. 2007). In addition, the CEA has an important role in the expression of fear CRs (Fendt and Fanselow 1999; LeDoux 2000; Davis and Whalen 2001; Maren 2001b; Fanselow and Gale 2003). In support of this, many studies have shown that either permanent or temporary lesions of the BLA or CEA prevent the acquisition and/or expression of fear memories (Helmstetter 1992; Helmstetter and Bellgowan 1994; Campeau and Davis 1995; Maren et al. 1996a,b; Killcross et al. 1997; Muller et al. 1997; Walker and Davis 1997; Cousens and Otto 1998; Maren 1998, 1999, 2001a,b; Wilensky et al. 1999, 2000, 2006; Goosens and Maren 2001, 2003; Nader et al. 2001; Fanselow and Gale 2003; Gale et al. 2004; Koo et al. 2004; Zimmerman et al. 2007).In addition to its role in encoding CS–US associations during conditioning, recent work suggests that the amygdala is also involved in representing properties of the US itself. For example, temporary or permanent lesions of the BLA reduce both decrements in conditioned responding after devaluation of a food US (Hatfield et al. 1996; Killcross et al. 1997; Blundell et al. 2001; Balleine et al. 2003; Everitt et al. 2003; Pickens et al. 2003; Holland 2004) and increments in conditional responding after inflation of a shock US (Fanselow and Gale 2003). Moreover, recent electrophysiological studies in primates indicate that amygdala neurons represent the value of both aversive and appetitive outcomes (Paton et al. 2006; Belova et al. 2007, 2008; Salzman et al. 2007). These studies suggest that one function of the BLA is to represent specific properties of biologically significant events, such as the food or shock USs that are typically used in Pavlovian conditioning paradigms. By this view, the BLA may represent specific sensory properties of USs that shape the nature of learned behavioral responses to the US (Balleine and Killcross 2006) and allow CSs to gain access to the incentive value of the US (Everitt et al. 2003).In contrast to this view, we recently reported that rats with neurotoxic BLA lesions exhibit normal US revaluation after Pavlovian fear conditioning (Rabinak and Maren 2008). In this study, auditory fear conditioning (75 CS–US trials) with a moderate footshock (1 mA) was followed by several exposures (five US-alone trials) to an intense footshock (3 mA) during an inflation session. Both intact rats and rats with BLA lesions exhibit a robust increase in conditional freezing to the auditory CS during a subsequent retention test (Rabinak and Maren 2008). Control experiments suggested that this was due to a revaluation of the US with which the CS was associated, rather than nonassociative sensitization of fear engendered by exposure to intense shock. These data reveal that the BLA may not be necessary for representing properties of shock USs during Pavlovian fear conditioning. To address these issues further, we have examined the consequence of reversible pharmacological manipulations of the amygdala during US inflation on conditional fear responses established with either extensive or limited training.     

Excitation and inhibition in unblocking

In four experiments the nature of learning established with unblocking procedures in the appetitive conditioning of rats was examined. Measures of learning included response topography, effects of selective satiation, and summation and retardation tests of conditioned inhibition. One cue (A) was first paired with either a single unconditioned stimulus event, US1, or a sequence of two events, US1----US2. US2 was either qualitatively similar to (US2-Same) or different from (US2-Diff) US1. Then, a compound of A and a novel cue (X) was reinforced with US1 or US1----US2. Conditioning to X was blocked if either the single US1 or the US1----US2 sequence was used in both phases. If X accompanied an upshift in the reinforcer, from US1 to US1----US2, it acquired conditioned responding, especially when US2-Diff was used. Responding in the latter case was the consequence of both X-US1 and X-US2 associations. In Experiments 1-3, if X accompanied a downshift from US1----US2-Same to US1, it acquired conditioned responding that was based on X-US1 associations, but if it accompanied a downshift from US1----US2-Diff to US1, it acquired conditioned inhibition based on X-US2 associations. In Experiment 4, X acquired net inhibition at short US1-US2 intervals and net excitation at longer intervals, with downshifts from either US1----US2-Same or US1----US2-Diff to US1. However, the interval gradient was broader with downshifts from US1----US2-Diff. These data, and several other contradictory findings in the unblocking literature, are consistent with the views that (a) the surprising addition or deletion of US2 in unblocking experiments both facilitates the acquisition of excitatory X-US1 associations and establishes either excitatory or inhibitory, respectively, X-US2 associations, and (b) that the gradient of X-US1 facilitation is broader than that of X-US2 association.