Ontogeny of eyeblink conditioning in the rat: Auditory frequency and discrimination learning effects

The present study sought to determine whether acoustic properties of the auditory conditioned stimulus (CS) or the use of a discrimination learning procedure would alter the emergence of eyeblink conditioning between postnatal Days 17 and 24 (Days 17–24) in the rat. In Experiment 1, we employed a 3 × 2 × 2 factorial design, involving pitch of the auditory CS (2.8, 5.0, and 9.0 kHz), training condition (paired vs. unpaired), and age (Days 17 or 24). Associative learning was evident at all tone frequencies on Day 24, but increased across frequencies on Day 17, although large age differences in conditioning remained at all tone frequencies. In Experiment 2, rat pups were trained to discriminate 2.8 versus 9.0-kHz tones on Day 17 or Day 24. Eyeblink conditioning increased with tone frequency on Day 24 but discriminative conditioning failed to appear on Day 17. These findings are discussed in relation to the role of auditory system maturation in the ontogeny of eyeblink conditioning. © 1995 Johnv Wiley & Sons, Inc.     

Ontogeny of eyeblink conditioning using a visual conditional stimulus

The developmental emergence of associative learning in rodents is determined by interactions among sensory, motor, and associative systems that are engaged in a particular experimental preparation (Carter & Stanton, 1996; Hunt & Campbell, 1997; Rudy, 1992). In fear conditioning, chemosensory, auditory, and visual cues emerge successively as effective conditional stimuli (CS) during postnatal ontogeny. In the present study, we begin to examine the generality of this principle of sensory system development for eyeblink conditioning, a form of associative learning that develops substantially later than conditioned fear (Carter & Stanton, 1996). We asked whether the developmental emergence of eyeblink conditioning to a visual CS occurs at an age that is the same or different from conditioning to an auditory CS. In Experiment 1, rat pups were trained on postnatal Day 17 or 24 with experimental parameters (and design) that were identical to our previous studies of eyeblink conditioning except that presentation of a light rather than a tone served as the CS. The outcome was also identical: no eyeblink conditioning on Day 17 and strong conditioning on Day 24. In Experiment 2, conditioning to tone versus light was directly compared by means of a discrimination learning design on postnatal Days 19, 21, 23, and 31. There was no evidence for differential development of auditory versus visual eyeblink conditioning. The difference between this outcome and previous ones involving conditioned fear (Hunt & Campbell, 1997; Rudy, 1992) suggests that principles concerning sensory maturation and learning may be different for early- versus late-developing associative systems.     

Ontogeny of delay versus trace eyeblink conditioning in the rat

The ontogeny of delay versus trace eyeblink conditioning was examined in 19-, 23-, and 30-day-old rat pups. Pairings of a tone conditioned stimulus (CS) and periocular shock unconditioned stimulus (US; 100-ms) were presented in one of three conditioning paradigms: standard delay [380-ms CS, 280-ms interstimulus interval (ISI)], trace (380-ms CS, 500-ms trace interval), or long-delay (980-ms CS, 880-ms ISI). The results of two experiments indicated that standard delay conditioning emerged between 19 and 23 days of age whereas trace and long-delay eyeblink conditioning emerged more slowly from postnatal Days 19 to 30. Because the acquisition profile for long-delay paralleled that of trace and not standard delay, it appears that the relative deficits in the emergence of trace eyeblink conditioning during development reflect difficulty in forming associations over long ISIs rather than the short-term memory demands of the trace conditioning paradigm.     

Early Pavlovian conditioning impairs later Pavlovian conditioning

Four experiments tested the effects in the rat of very early experience with stimuli to be used later for Pavlovian conditioning. Beginning on postnatal Day 12, prior to the development of substantial detection and effective perception of visual and auditory stimuli, rats were given five daily experiences with either lights or tones and a footshock known to be an effective unconditioned stimulus at these ages. Twenty-four hours after the last of these experiences, pairings of either the light or tone and the unconditioned stimulus were given with parameters established to yield a moderate degree of conditioning in untreated preweanlings (Experiment 1). Experiment 2 determined that early experience with paired or unpaired presentations of either the light or tone and the unconditioned stimulus resulted in a failure to condition to these same lights or tones on postnatal Day 17, although nontreated pups from the same litters conditioned quite effectively. Experiment 3 determined that this early conditioning experience with either paired or unpaired presentations of the lights or tones and the unconditioned stimulus yielded impaired conditioning on postnatal Day 17 in the alternative sensory modality as well, although again nontreated siblings conditioned quite effectively. Experiment 4 replicated the results of each of Experiments 2 and 3 and determined in addition that despite the impairment in conditioning that resulted from early paired or unpaired experience with the stimuli of conditioning, early experience with the individual stimuli of conditioning—with only the CS, the US, or the context—did not result in a similar impairment in conditioning. Although the results were unexpected, they may be understood in part in terms of intersensory competition during development, and there is precedent in the literature for similar interfering effects of early learning on later learning in a variety of species. © 1996 John Wiley & Sons, Inc.     

CS-US preexposure effects on trace eyeblink conditioning in young rats: potential implications for functional brain development

Recent studies of delay eyeblink conditioning (EBC) in young rats have demonstrated different effects of various conditioned and unconditioned stimulus (CS-US) preexposure conditions on learning at different ages. The present study extends this research to trace EBC. Subjects experienced 1 of 3 preexposure conditions (paired CS-US, unpaired CS-US, or no stimuli) at either 20 or 24 days of age. Four days later, they were conditioned using either trace (Experiment 1) or delay (Experiment 2) EBC parameters. Results were similar at both ages tested. Paired preexposure facilitated acquisition of delay but not trace relative to context preexposure. Unpaired preexposure impaired acquisition of both delay and trace. These behavioral findings provide a foundation for hypotheses about the functional maturation of cerebellar, hippocampal, and entorhinal learning circuits.     

Eyeblink conditioning using cochlear nucleus stimulation as a conditioned stimulus in developing rats

Previous studies demonstrated that the development of auditory conditioned stimulus (CS) input to the cerebellum may be a neural mechanism underlying the ontogenetic emergence of eyeblink conditioning in rats. The current study investigated the role of developmental changes in the projections of the cochlear nucleus (CN) in the ontogeny of eyeblink conditioning using electrical stimulation of the CN as a CS. Rat pups were implanted with a bipolar stimulating electrode in the CN and given six 100-trial training sessions with a 300 ms stimulation train in the CN paired with a 10 ms periorbital shock unconditioned stimulus (US) on postnatal days (P) 17-18 or 24-25. Control groups were given unpaired presentations of the CS and US. Rats in both age groups that received paired training showed significant increases in eyeblink conditioned responses across training relative to the unpaired groups. The rats trained on P24-25, however, showed stronger conditioning relative to the group trained on P17-18. Rats with missed electrodes in the inferior cerebellar peduncle or in the cerebellar cortex did not show conditioning. The findings suggest that developmental changes in the CN projections to the pons, inferior colliculus, or medial auditory thalamus may be a neural mechanism underlying the ontogeny of auditory eyeblink conditioning.     

Ontogeny of eyeblink conditioned response timing in rats

Eyeblink conditioned response (CR) timing was assessed in adult and infant rats. In Experiment 1, adult rats were trained with a 150-ms tone conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US; presented at 200- or 500-ms interstimulus intervals [ISIs]). The rats acquired CRs with 2 distinct peaks that occurred just before the US onset times. Experiments 2 and 3 examined developmental changes in CR timing in pups trained on Postnatal Days 24-26 or 32-34. Experiment 3 used a delay conditioning procedure in which the tone CS continued throughout the ISIs. Pups of both ages exhibited robust conditioning. However, there were age-related increases in the percentage of double-peaked CRs and in CR timing precision. Ontogenetic changes in eyeblink CR timing may be related to developmental changes in cerebellar cortical or hippocampal function.     

Conditioned facilitation of the unconditioned reflex after classical eyeblink conditioning.

The present study (N=40) investigated the time-course of conditioned facilitation of the unconditioned eyeblink reflex (UR). In a single-cue delay classical conditioning procedure, a tone conditioned stimulus (CS) signaled an airpuff unconditioned stimulus (airpuff US) to the eye. A paired group received 40 trials of CS/US presentations with an interstimulus interval (ISI) of 200 ms. An unpaired group received an equal number of explicitly unpaired presentations of the CS and US. Thereafter, eyeblink reflex facilitation was assessed by presenting 94 dB white noise (noise US) 10, 30, 50, 100, 150, and 1000 ms after CS onset. In the paired group, URs were significantly increased as early as 100 ms after CS onset compared to the unpaired group. This reflex facilitation was correlated with CR magnitude, indicating that conditioned facilitation of eyeblink URs indexes an early, automatic, preattentive stage in CR formation.     

Associative and non-associative blinking in classically conditioned adult rats

Over the last several years, a growing number of investigators have begun using the rat in classical eyeblink conditioning experiments, yet relatively few parametric studies have been done to examine the nature of conditioning in this species. We report here a parametric analysis of classical eyeblink conditioning in the adult rat using two conditioned stimulus (CS) modalities (light or tone) and three interstimulus intervals (ISI; 280, 580, or 880 ms). Rats trained at the shortest ISI generated the highest percentage of conditioned eyeblink responses (CRs) by the end of training. At the two longer ISIs, rats trained with the tone CS produced unusually high CR percentages over the first few acquisition sessions, relative to rats trained with the light CS. Experiment 2 assessed non-associative blink rates in response to presentations of the light or tone, in the absence of the US, at the same ISI durations used in paired conditioning. Significantly more blinks occurred with longer than shorter duration lights or tones. A higher blink rate was also recorded at all three durations during the early tone-alone sessions. The results suggest that early in classical eyeblink conditioning, rats trained with a tone CS may emit a high number of non-associative blinks, thereby inflating the CR frequency reported at this stage of training.     

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.     

Novel factors contributing to the expression of latent inhibition

Behavioral and neural correlates of latent inhibition (LI) during eyeblink conditioning were studied in 2 experiments. In Experiment 1, rabbits (Oryctolagus cuniculus) were conditioned after 8 days of tone conditioned stimulus (CS) presentations or 8 days of context-alone experience. LI was seen in the CS-preexposed rabbits when a relatively intense (5 psi) airpuff unconditioned stimulus was paired with the CS. In Experiment 2, rabbits were given 0, 4, or 8 days of CS preexposures or context-alone experience. Hippocampal activity was monitored from the 8-day CS- or context-exposure rabbits. The LI effect was seen only in rabbits given 4 days of CS preexposure, thus suggesting that LI depended largely on the rate of acquisition in the context-preexposed control group. The neural recordings showed that the hippocampus was sensitive to the relative novelty of the stimuli and the overall context, regardless of whether exposure to stimuli and context promoted LI.     

Amygdala lesions block conditioned enhancement of the early component of the rat eyeblink reflex

A tone conditioned stimulus (CS) previously paired with a grid shock unconditioned stimulus (US) can greatly enhance the early electromyographic (EMG) component (R1) of the rat eyeblink reflex. The hypothesis that the central nucleus of the amygdala (ACe) is an essential part of the circuitry mediating conditioned R1 enhancement was tested. After bilateral ACe lesions (L) or a sham operation (S), rats received paired presentations of the CS and US (P) or explicitly unpaired CS and US presentations (U), resulting in 4 groups: P/S, P/L, U/S, and U/L. ACe lesions completely prevented conditioned R1 enhancement, which was only exhibited in Group P/S. In the latter group, the "preextinction" conditioned enhancement effect was roughly a 2-fold increase in the R1 magnitude. Circuit-level mechanisms are discussed, and some advantages of the eyeblink EMG response in this general conditioning paradigm are considered.     

Effects of Conditioned Stimulus Fear-Relevance and Preexposure on Expectancy and Electrodermal Measures of Human Pavlovian Conditioning

The present research investigated the effects of fear-relevance of the conditioned stimulus (CS) and CS preexposure on human electrodermal conditioning and on a continuous measure of expectancy of the unconditioned stimulus (US). Both experiments employed 20 preexposure, 8 acquisition, and 8 extinction trials in a differential Pavlovian conditioning paradigm with shock as the US. In Experiment 1 (N = 48), electrodermal conditioning was retarded by CS preexposure, but was not influenced by fear-relevance of the CS. Expectancy of the US was retarded by preexposure only in the fear-relevant condition. In Experiment 2 (N = 48), the CS/US contingencies was embedded in a visual masking task. Preexposure retarded both electrodermal conditioning and US expectancy. Neither measure was influenced by fear-relevance of the CS. However, fewer subjects in the preexposure condition learned the CS/US relationship and those who did, did so on later trial than those in the no-preexposure condition. Thus, the results indicated clear retardation of conditioning as a result of preexposure, but no reliable effect of fear-relevance.     

Activity profiles of single neurons in caudal anterior cingulate cortex during trace eyeblink conditioning in the rabbit

Acquisition of trace eyeblink conditioning involves the association of a conditioned stimulus (CS) with an unconditioned stimulus (US) separated by a stimulus-free trace interval. This form of conditioning is dependent upon the hippocampus and the caudal anterior cingulate cortex (AC), in addition to brain stem and cerebellar circuitry. Hippocampal involvement in trace eyeblink conditioning has been studied extensively, but the involvement of caudal AC is less well understood. In the present study, we compared neuronal responses from rabbits given either paired (trace conditioning) or unpaired (pseudoconditioning) presentations of the CS and US. Presentation of the CS elicited significant increases in neuronal activity at the onset of both trace conditioning and pseudoconditioning. A robust CS-elicited neuronal response persisted throughout the first 2 days of trace conditioning, declining gradually across subsequent training sessions. In contrast, the magnitude of the CS-elicited excitatory response during pseudoconditioning began to decline within the first 10 trials. Neurons exhibiting excitatory responses to the CS during trace conditioning also exhibited excitatory responses to the US that were significantly greater in magnitude than US-elicited responses during pseudoconditioning. CS-elicited decreases in neuronal activity became more robust over the course of trace conditioning compared to pseudoconditioning. Reductions in activity during the CS interval consistently preceded excitation in both training groups, suggesting that the CS-elicited decreases in neuronal activity may serve to increase the signal-to-noise ratio of the excitatory response to the tone. Taken together, these data suggest that the caudal AC is involved early in trace eyeblink conditioning and that maintenance of the CS-elicited excitatory response may serve to signal the salience of the tone.     

Classical conditioning in the rat fetus: involvement of mu and kappa opioid systems in the conditioned response

When the Embryonic Day 20 (E20) rat fetus is given a conditioning trial involving a paired presentation of an artificial nipple (the conditioned stimulus; CS) with an intraoral infusion of milk (the unconditioned stimulus; US), it shows evidence of classical conditioning when again exposed to the CS during a test trial. Specifically, the fetus shows fewer oral grasp responses (the conditioned response; CR) when continuously presented with the artificial nipple. The present study further investigated this classically conditioned reduction in oral grasping. Separate experiments (a) examined the time course of the reduction in oral grasping (Experiment 1), (b) characterized the time course of mu opioid (Experiment 2) and kappa opioid (Experiment 3) involvement in the CR, and (c) described changes in fetal behavior (Experiment 4) associated with mu and kappa opioid effects on responding to the artificial nipple. The results are discussed in terms of opioid involvement in establishing and maintaining early suckling behavior.     

The effect of emotional and attentional load on attentional startle modulation

The interactive effects of emotion and attention on attentional startle modulation were investigated in two experiments. Participants performed a discrimination and counting task with two visual stimuli during which acoustic eyeblink startle-eliciting probes were presented at long lead intervals. In Experiment 1, this task was combined with aversive Pavlovian conditioning. In Group Attend CS+, the attended stimulus was followed by an aversive unconditional stimulus (US) and the ignored stimulus was presented alone whereas the ignored stimulus was paired with the US in Group Attend CS−. In Experiment 2, a non-aversive reaction time task US replaced the aversive US. Regardless of the conditioning manipulation and consistent with a modality non-specific account of attentional startle modulation, startle magnitude was larger during attended than ignored stimuli in both experiments. Blink latency shortening was differentially affected by the conditioning manipulations suggesting additive effects of conditioning and discrimination and counting task on blink startle.     

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.     

Classical conditioning in the fetal rat with a long delay between presentation of CS and US

A single paired presentation of the artificial nipple and milk results in classical conditioning of changes in perioral responsiveness in the E20 rat fetus. This classical conditioning is evidenced by a reduction in responding to perioral tactile stimulation. The results of Experiment 1 confirmed the specificity of milk as an unconditioned stimulus to support classical conditioning. Experiment 2 demonstrated that single-trial classical conditioning with the artificial nipple CS and milk US was possible with a delay of 30 s between nipple and milk presentations. Further, measurements of fetal motor behavior during the delay between CS and US presentations indicated that a single 15-s presentation of the artificial nipple increased movements of the mouth for 30 s after removal of the artificial nipple. Experiment 3 demonstrated that three exposures to the artificial nipple prolonged the expression of mouthing for up to 120 s and made possible single trial classical conditioning with a delay of 120 s between CS and US presentations. The capacity of the fetal CNS to maintain a “behavioral trace” for an ecologically important stimulus, such as the nipple, could have adaptive significance in the early development of motivated behavior. © 1997 John Wiley & Sons, Inc. Dev Psychobiol 30: 49–59, 1997     

Reflex facilitation during eyeblink conditioning and subsequent interpositus nucleus inactivation in the rabbit (Oryctolagus cuniculus)

In eyeblink conditioning in the rabbit (Oryctolagus cuniculus), not only is a conditioned response (CR) acquired, but also the original reflex is modified as a function of training. In Experiment 1, by comparing unconditioned responses in unpaired and paired groups, 3 types of reflex facilitation were distinguished. One type was linked to exposure to the unconditioned stimuli (USs) and/or experimental setting. The 2nd type was related to the formation of the memory trace for conditioned eyeblink. The 3rd type was linked to the conditioned stimulus immediately preceding the US in the paired group. In Experiment 2, reversible inactivation of the interpositus nucleus (IPN) abolished the CR and reduced the CR-related reflex facilitation, indicating that the latter depends on the plasticity of the IPN.     

Conditioning of aversion to an odor paired with peripheral shock in the developing rat

Four experiments examined an apparent inability to associate, or severe deficiency in associating, an odor and a footshock during the first 2 weeks of life in the rat, a cue-to-consequence relationship that had formerly seemed age-dependent. With a particular classical conditioning procedure, however, significiant conditioning occurred on postnatal Days 6 and 10 with relatively few conditioning trials; the procedure employed an odor explicitly unpaired with footshock (CS?), as well as an odor paired with footshock (CS+) (Experiment I). Experiment II assessed the contribution of a CS? exposure in the conditioning of rats 8, 15, or 50 days of age. For 8-day-olds, exposure to both the CS+ and CS? resulted in conditioned aversion to the CS+ after eight but not one conditioning trials, but neither 1 nor eight trials with only a CS+ produced conditioning. For 15- and 50-day-olds, conditioning to the CS+ odor was significant after one trial with, but not without, a specific CS? with eight trials, however, conditioning was significant with or without the specific CS?. It was verified with the 50-day-olds in Experiment III that aversion to the CS+ was conditioned with a single trial only if a CS? had been presented, with a slight trend toward superior conditioning if the CS? preceded rather than followed the CS+ during conditioning. Experiment IV tested the hypothesis that exposure to the distinctive CS? odor sensitzes the animal to the specific properties of the CS+ odor. Fifteen and 50-day-old rats were given one conditioning trial with a CS+ odor that was either unaccompanied by a CS? or that was presented with a CS? odor in the same context as the CS+ or in a different context. For both 15? and 50-day-old rats, conditioning to the CS+ occurred only for animals given the CS? in the same context as the CS+, indicating that the hypothesis should be rejected. The results generally indicate rapid and substantial odor-footshock conditioning in rats as young as 6 days of age, with CS-exposure established as perhaps especially significant for conditioning early in life, but important for rats of all ages tested, from infancy to adulthood.