Adolescent exposure to fluoxetine impairs serial pattern learning in the serial multiple choice (SMC) task in adult rats.

The effects of chronic adolescent fluoxetine (FLX, Prozac®) exposure on adult cognition are largely unknown. We used a serial multiple choice (SMC) task to characterize the effects of adolescent FLX exposure on rat serial pattern learning in adulthood. Male rats were exposed to either 1.0, 2.0, or 4.0 mg/kg/day FLX for five consecutive days each week for five weeks during adolescence, followed by a 35-day drug-free period. As adults, the rats were trained in a task that required them to learn a highly structured sequential pattern of responses in an octagonal chamber for water reinforcement. In a transfer phase, the terminal element of the pattern was replaced by a violation element that was inconsistent with previously learned pattern structure. Results indicated that adolescent FLX exposure caused differential learning deficits for different types of elements in the serial pattern. Adolescent exposure to 1.0 or 4.0 mg/kg/day FLX, but not 2.0 mg/kg/day FLX, impaired chunk-boundary element learning, which is known to be mediated by stimulus-response (S-R) learning. All three doses of FLX impaired violation element learning, which is known to be mediated by multiple-cue learning. FLX did not impair within-chunk element learning, which is known to be mediated by rule-learning mechanisms. The results indicate that adolescent FLX exposure produced multiple cognitive impairments that were detectable in adulthood long after drug exposure ended.

Serial pattern retention in male and female rats.

Serial pattern learning is a model paradigm for studying parallel-processing in complex learning in rats. The current experiment extends the paradigm to the study of sequential memory by examining forgetting curves for the component element types that make up a serial pattern. Adult male and female rats were trained in a serial multiple choice (SMC) task in which rats learned a serial pattern of nose-poke responses in a circular array of 8 receptacles mounted on the walls of an octagonal operant chamber. The pattern was 123-234-345-456-567-678-781-818, where digits represent the clockwise positions of successive correct receptacles. Previous work has shown that chunk-boundary elements (the first element of each 3-element chunk), within-chunk elements (the second and third elements in all but the last chunk), and the violation element (the last element of the pattern) are learned via different cognitive mechanisms. After each rat was trained to an 85% correct performance criterion on the violation element, we then assessed serial pattern retention at 24-h, 2-week, and 4-week retention intervals. For chunk-boundary and within-chunk elements, forgetting was observed only at the 4-week retention interval. Sex differences were observed; females performed better than males on within-chunk elements at 24-h and 4-week retention intervals. For the violation element, significant forgetting was observed earlier at the 2-week retention interval as well as at the 4-week retention interval. Thus, pattern elements that were learned slower were forgotten faster. The experiment provides a proof of concept for evaluating forgetting curves separately for the multiple memory systems rats appear to employ concurrently in this paradigm, a method that may prove useful in characterizing the impact of relevant neurobiological manipulations on forgetting in multiple sequential memory systems.

Strategy breakdown following muscarinic blockade in rats.

The present study examined the effects of systemically administered atropine sulfate, a muscarinic cholinergic antagonist, on a series of probe tests in the retention of a highly-structured serial pattern in a serial multiple choice (SMC) task. Rats were trained on a 24-element pattern composed of eight 3-element chunks ending with a violation element: 123-234-345-456-567-678-781-818 where the digits represent the clockwise position of levers in an octagonal chamber, dashes indicate 3-s pauses termed "phrasing cues," and other intertrial intervals were 1s. In daily acquisition trials rats were given either 50mg/kg of atropine sulfate or an equivalent amount of saline (Chenoweth & Fountain, 2015). Following acquisition, rats were given a series of drug challenges, and the present study reports a series of Phrasing Cue Removal Probes that tested rats' abilities to make correct responses in the absence of phrasing cues. Rats tested under atropine demonstrated more difficulty in recalling encoded responses in these probe trials than did rats tested under saline. The results indicate that intact central muscarinic cholinergic systems were needed for rats to display efficient adaptive response strategies under conditions where some features of the previously-learned pattern change.

Central muscarinic cholinergic involvement in serial pattern learning: Atropine impairs acquisition and retention in a serial multiple choice (SMC) task in rats.

Atropine sulfate is a muscarinic cholinergic antagonist which impairs acquisition and retention performance on a variety of cognitive tasks. The present study examined the effects of atropine on acquisition and retention of a highly-structured serial pattern in a serial multiple choice (SMC) task. Rats were given daily intraperitoneal injections of either saline or atropine sulfate (50mg/kg) and trained in an octagonal operant chamber equipped with a lever on each wall. They learned to press the levers in a particular order (the serial pattern) for brain-stimulation reward in a discrete-trial procedure with correction. The two groups learned a pattern composed of eight 3-element chunks ending with a violation element: 123-234-345-456-567-678-781-818 where the digits represent the clock-wise positions of levers in the chamber, dashes indicate 3-s pauses, and other intertrial intervals were 1s. Central muscarinic cholinergic blockade by atropine caused profound impairments during acquisition, specifically in the encoding of chunk-boundary elements (the first element of chunks) and the violation element of the pattern, but had a significant but negligible effect on the encoding of within-chunk elements relative to saline-injected rats. These effects persisted when atropine was removed, and similar impairments were also observed in retention performance. The results indicate that intact central muscarinic cholinergic systems are necessary for learning and producing appropriate responses at places in sequences where pattern structure changes. The results also provide further evidence that multiple cognitive systems are recruited to learn and perform within-chunk, chunk-boundary, and violation elements of a serial pattern.

Central cholinergic involvement in sequential behavior: impairments of performance by atropine in a serial multiple choice task for rats.

Two experiments examined whether muscarinic cholinergic systems play a role in rats' ability to perform well-learned highly-structured serial response patterns, particularly focusing on rats' performance on pattern elements learned by encoding rules versus by acquisition of stimulus-response (S-R) associations. Rats performed serial patterns of responses in a serial multiple choice task in an 8-lever circular array for hypothalamic brain-stimulation reward. Two experiments examined the effects of atropine, a centrally-acting muscarinic cholinergic receptor antagonist, on rats' ability to perform pattern elements where responses were controlled by rules versus elements, such as rule-inconsistent "violation elements" and elements following "phrasing cues," where responses were controlled by associative cues. In Experiment 1, 3-element chunks of both patterns were signaled by pauses that served as phrasing cues before chunk-boundary elements, but one pattern also included a violation element that was inconsistent with pattern structure. Once rats reached a high criterion of performance, the drug challenge was intraperitoneal injection of a single dose of 50 mg/kg atropine sulfate. Atropine impaired performance on elements learned by S-R learning, namely, chunk-boundary elements and the violation element, but had no effect on performance of rule-based within-chunk elements. In Experiment 2, patterns were phrased and unphrased perfect patterns (i.e., without violation elements). To control for peripheral effects of atropine, rats were treated with a series of doses of either centrally-acting atropine or peripherally-acting atropine methyl nitrate (AMN), which does not cross the blood-brain barrier. Once rats reached a high criterion, the drug challenges were on alternate days in the order 50, 25, and 100 mg/kg of either atropine sulfate or AMN. Atropine, but not AMN, impaired performance in the phrased perfect pattern for pattern elements where S-R associations were important for performance, namely, chunk-boundary elements. However, in the structurally more ambiguous unphrased perfect pattern where rats had fewer cues and presumably relied more on S-R associations throughout, atropine impaired performance on all pattern elements. Thus, intact muscarinic cholinergic systems were shown to be necessary for discriminative control previously established by S-R learning, but were not necessary for rule-based serial pattern performance.

Irrelevant relations and the active search for pattern structure in rat serial pattern learning.

Hersh (Mem Cogn 2:771-774, 1974) investigated the role of irrelevant relations in college students' pattern learning and performance for letter series completion problems. He created irrelevant relations in sequences by inserting items to make pattern structure ambiguous such that it was open to multiple interpretations during initial pattern processing. He reported irrelevant relations impaired humans' performance more when placed at the beginning of patterns than at the end. However, once pattern structure was induced, irrelevant relations were not impairing. Here, we examined the impact on rat serial pattern learning of irrelevant relations positioned at the beginning or end of a serial pattern. Rats pressed levers in a circular array according to the same structured serial pattern, 123 234 345 456 567, where digits indicated the clockwise position of the correct lever. This structured serial pattern was interleaved with repeating responses on lever 2 to produce irrelevant relations at the beginning of the pattern (Beginning: 122232 223242 324252 425262 526272), on lever 6 to produce irrelevant relations at the end of the pattern (End: 162636 263646 364656 465666 566676), or on lever 8 to produce no irrelevant relations (No Irrelevant Relations: 182838 283848 384858 485868 586878. Irrelevant relations significantly retarded learning regardless of their placement within the pattern. However, irrelevant relations retarded learning significantly more when placed at the pattern beginning versus end. The results indicate that rats, like humans, process patterns from beginning to end.

Rats' use of hierarchical organization in serial pattern learning.

Many organisms show aptitude for learning and performing patterned sequences. However, we do not yet have a complete account of how they accomplish this. One of the most successful is Restle's hierarchical model, which supposes organisms represent sequences using the simplest form available through using hierarchies to organize the sequences' elements. In two experiments, we evaluated if Restle's model accurately accounted for rats' patterned sequence learning. First, we compared rats' learning of a runs pattern with three levels of hierarchical organization with no violations of pattern structure to learning of the same pattern with one violation of pattern structure in the pattern's second half. Restle's model predicts rats should have more difficulty learning the violation pattern and that rats learning the pattern with the violation in the second half of the pattern should attempt to reflect that violation to the first half. The results indicated that rats had more difficulty learning the violation pattern and that inserting a violation in the second half of the pattern led rats to make significantly more errors on the second half of the pattern relative to the first half of the pattern. Rats did not insert the violation from the second half of the pattern into the first half of the pattern. In a second experiment, we continued to explore if Restle's model accurately accounted for rats' pattern learning using more complex patterns. The present results of both experiments and prior work with humans were inconsistent with Restle's model, suggesting more work is needed to develop a model that accurately accounts for rats' learning of complex patterned sequences.

Sex differences in serial pattern learning in mice.

Numerous investigators have examined the hypothesis that males and females learn or perform differentially on various tasks. However, many of the behavioural investigations with nonhuman animals (e.g., rats) have used paradigms that do not permit the exploration of complex learning and memory between the sexes. To this end, we explored the ability of male versus female mice to learn three different patterns in succession in three separate experiments: single alternation (e.g., right, left, right, left), double alternation (e.g., right, right, left, left), and runs (e.g., 123, 234, 345, 456, 567, 678, 781, 812, where digits represent locations within a circular array in the counterclockwise direction). We hypothesized that sex differences, if they existed, would be most likely to appear as the pattern to be learned became more complex (required more rules to capture how elements relate to one another). The results indicated that mice can learn all three pattern types, but learning was more difficult as pattern complexity increased. Males learned the runs pattern significantly more quickly than females did; no significant differences were found between males and females for acquisition of the single-alternation or double-alternation patterns. These results suggest that sex differences in serial pattern learning within rodents are not unique to rats and are more likely to be seen during acquisition of more complex patterns.

Adolescent exposure to nicotine impairs adult serial pattern learning in rats.

In the present study investigating the effects of adolescent nicotine exposure on adult serial pattern learning, adolescent rats received daily i.p. injections of either 1.0 mg/kg nicotine or saline for 5 days per week for 5 weeks beginning on postnatal day 25 (P25), then were allowed 35 days drug free. Rats then began training on P95 as adults on a 24-element serial pattern composed of eight 3-element chunks. Adolescent exposure to 1.0 mg/kg nicotine produced persistent retardation of learning for the first element of each 3-element chunk of the pattern, that is, for chunk boundary elements, and transient retardation of learning for elements 2 and 3 of each chunk of the pattern, that is, for the within-chunk elements. Deficits at chunk boundaries were interpreted as deficits of phrasing cue discrimination learning whereas deficits for learning responses for elements within-chunks (elements 2 and 3 of chunks) were interpreted as deficits of rule learning. These results indicate that the effects of adolescent nicotine exposure on adult learning and cognitive capacity deserve further scrutiny.

Serial pattern learning in pigeons: Rule-based or associative?

Extensive research has documented evidence for rule learning in sequential behavior tasks in both rats and humans. We adapted the 2-choice serial multiple choice (SMC) task developed for use with rats (Fountain & Rowan, 1995a) to study sequence behavior in pigeons. Pigeons were presented with 8 disks arranged in a circular array on a touchscreen, and pecking to an illuminated disk could lead to reward. Correct responding consisted of serial patterns involving "run" chunks of 3 elements (123 234, etc.). Some pigeons experienced a violation of the chunk rule in the final chunk. Unlike rats, pigeons made fewer errors on violation chunks than run chunks, suggesting the use of low-level cues to guide choices. Removal of low-level cues and increasing the number of simultaneously illuminated disks to an 8-choice SMC task resulted in more errors on the violation chunk. Pigeons were able to use the rule when the array of disks was contracted or expanded, and when chunk length was extended to 4 and 5 elements, but not when disks were removed from or added to the array. Pigeons were also able to abstract structure from a "trill" pattern (121 232 etc.), as shown by high error rates on a violation trial. These results suggest that pigeons, like rats and humans, can abstract sequence structure, but do so primarily in the absence of specific low-level feature-based information. (PsycINFO Database Record

Transgenerational effects of adolescent nicotine exposure in rats: Evidence for cognitive deficits in adult female offspring.

This study investigated whether adolescent nicotine exposure in one generation of rats would impair the cognitive capacity of a subsequent generation. Male and female rats in the parental F0 generation were given twice-daily i.p. injections of either 1.0mg/kg nicotine or an equivalent volume of saline for 35days during adolescence on postnatal days 25-59 (P25-59). After reaching adulthood, male and female nicotine-exposed rats were paired for breeding as were male and female saline control rats. Only female offspring were used in this experiment. Half of the offspring of F0 nicotine-exposed breeders and half of the offspring of F0 saline control rats received twice-daily i.p. injections of 1.0mg/kg nicotine during adolescence on P25-59. The remainder of the rats received twice-daily saline injections for the same period. To evaluate transgenerational effects of nicotine exposure on complex cognitive learning abilities, F1 generation rats were trained to perform a highly structured serial pattern in a serial multiple choice (SMC) task. Beginning on P95, rats in the F1 generation were given either 4days of massed training (20patterns/day) followed by spaced training (10 patterns/day) or only spaced training. Transgenerational effects of adolescent nicotine exposure were observed as greater difficulty in learning a "violation element" of the pattern, which indicated that rats were impaired in the ability to encode and remember multiple sequential elements as compound or configural cues. The results indicated that for rats that received massed training, F1 generation rats with adolescent nicotine exposure whose F0 generation parents also experienced adolescent nicotine exposure showed poorer learning of the violation element than rats that experienced adolescent nicotine exposure only in the F1 generation. Thus, adolescent nicotine exposure in one generation of rats produced a cognitive impairment in the next generation.

Concurrent cognitive processes in rat serial pattern learning: II. Discrimination learning, rule learning, chunk length, and multiple-item memories.

The current experiment examined the factors that determine acquisition for elements of highly structured serial patterns. Three groups of rats were trained on three patterns with parallel rule-based hierarchical structure, but with 3-, 4-, or 5-element chunks, each with a final violation element. Once rats mastered their patterns, probe patterns were introduced to answer several questions. To assess the extent to which the learned response pattern depended on intrachamber location cues for anticipating different element types, Spatial Shift Probes shifted the starting lever of patterns to locations that positioned chunk boundaries where they had never been experienced during training. To assess the extent to which a phrasing cue is necessary for rats to perform a chunk-boundary response, a Cue Removal Probe tested whether rats would produce a chunk-boundary response in the correct serial position if the phrasing cue was omitted. To assess the extent to which cues from multiple trials leading up to the violation element are required to anticipate the violation element, Multiple-Item Memory Probes required rats to make an unexpected response on one of the elements in the last two chunks of the pattern prior to the violation element. The results indicated that rats used multiple concurrent learning and memory processes to master serial patterns, including discrimination learning, rule learning, encoding of chunk length, and multiple-item memories.

Paradoxical effects of injection stress and nicotine exposure experienced during adolescence on learning in a serial multiple choice (SMC) task in adult female rats.

Nicotine exposure in adolescent rats has been shown to cause learning impairments that persist into adulthood long after nicotine exposure has ended. This study was designed to assess the extent to which the effects of adolescent nicotine exposure on learning in adulthood can be accounted for by adolescent injection stress experienced concurrently with adolescent nicotine exposure. Female rats received either 0.033 mg/h nicotine (expressed as the weight of the free base) or bacteriostatic water vehicle by osmotic pump infusion on postnatal days 25-53 (P25-53). Half of the nicotine-exposed rats and half of the vehicle rats also received twice-daily injection stress consisting of intraperitoneal saline injections on P26-53. Together these procedures produced 4 groups: No Nicotine/No Stress, Nicotine/No Stress, No Nicotine/Stress, and Nicotine/Stress. On P65-99, rats were trained to perform a structurally complex 24-element serial pattern of responses in the serial multiple choice (SMC) task. Four general results were obtained in the current study. First, learning for within-chunk elements was not affected by either adolescent nicotine exposure, consistent with past work (Pickens, Rowan, Bevins, and Fountain, 2013), or adolescent injection stress. Thus, there were no effects of adolescent nicotine exposure or injection stress on adult within-chunk learning typically attributed to rule learning in the SMC task. Second, adolescent injection stress alone (i.e., without concurrent nicotine exposure) caused transient but significant facilitation of adult learning restricted to a single element of the 24-element pattern, namely, the "violation element," that was the only element of the pattern that was inconsistent with pattern structure. Thus, adolescent injection stress alone facilitated violation element acquisition in adulthood. Third, also consistent with past work (Pickens et al., 2013), adolescent nicotine exposure, in this case both with and without adolescent injection stress, caused a learning impairment in adulthood for the violation element in female rats. Thus, adolescent nicotine impaired adult violation element learning typically attributed to multiple-item learning in the SMC task. Fourth, a paradoxical interaction of injection stress and nicotine exposure in acquisition was observed. In the same female rats in which violation-element learning was impaired by adolescent nicotine exposure, adolescent nicotine experienced without adolescent injection stress produced better learning for chunk-boundary elements in adulthood compared to all other conditions. Thus, adolescent nicotine without concurrent injection stress facilitated adult chunk-boundary element learning typically attributed to concurrent stimulus-response discrimination learning and serial-position learning in the SMC task. To the best of our knowledge, the current study is the first to demonstrate facilitation of adult learning caused by adolescent nicotine exposure.

Adolescent exposure to methylphenidate impairs serial pattern learning in the serial multiple choice (SMC) task in adult rats.

The long-term effects of adolescent exposure to methylphenidate (MPD) on adult cognitive capacity are largely unknown. We utilized a serial multiple choice (SMC) task, which is a sequential learning paradigm for studying complex learning, to observe the effects of methylphenidate exposure during adolescence on later serial pattern acquisition during adulthood. Following 20.0mg/kg/day MPD or saline exposure for 5 days/week for 5 weeks during adolescence, male rats were trained to produce a highly structured serial response pattern in an octagonal operant chamber for water reinforcement as adults. During a transfer phase, a violation to the previously-learned pattern structure was introduced as the last element of the sequential pattern. Results indicated that while rats in both groups were able to learn the training and transfer patterns, adolescent exposure to MPD impaired learning for some aspects of pattern learning in the training phase which are learned using discrimination learning or serial position learning. In contrast adolescent exposure to MPD had no effect on other aspects of pattern learning which have been shown to tap into rule learning mechanisms. Additionally, adolescent MPD exposure impaired learning for the violation element in the transfer phase. This indicates a deficit in multi-item learning previously shown to be responsible for violation element learning. Thus, these results clearly show that adolescent MPD produced multiple cognitive impairments in male rats that persisted into adulthood long after MPD exposure ended.

What is learned in sequential learning? An associative model of reward magnitude serial-pattern learning.

A computational model of sequence learning is described that is based on pairwise associations and generalization. Simulations by the model predicted that rats should learn a long monotonic pattern of food quantities better than a nonmonotonic pattern, as predicted by rule-learning theory, and that they should learn a short nonmonotonic pattern with highly discriminable elements better than 1 with less discriminable elements, as predicted by interitem association theory. In 2 other studies, the model also simulated behavioral "rule generalization," "extrapolation," and associative transfer data motivated by both rule-learning and associative perspectives. Although these simulations do not rule out the possibility that rats can use rule induction to learn serial patterns, they show that a simple associative model can account for the classical behavioral studies implicating rule learning in reward magnitude serial-pattern learning.

Sex differences in adult cognitive deficits after adolescent nicotine exposure in rats.

This study was designed to determine whether deficits in adult serial pattern learning caused by adolescent nicotine exposure persist as impairments in asymptotic performance, whether adolescent nicotine exposure differentially retards learning about pattern elements that are inconsistent with "perfect" pattern structure, and whether there are sex differences in rats' response to adolescent nicotine exposure as assessed by a serial multiple choice task. The current study replicated the results of our initial report (Fountain et al., 2008) using this task by showing that adolescent nicotine exposure (1.0mg/kg/day nicotine for 35days) produced a specific cognitive impairment in male rats that persisted into adulthood at least a month after adolescent nicotine exposure ended. In addition, sex differences were observed even in controls, with additional evidence that adolescent nicotine exposure significantly impaired learning relative to same-sex controls for chunk boundary elements in males and for violation elements in females. All nicotine-induced impairments were overcome by additional training so that groups did not differ at asymptote. An examination of the types of errors rats made indicated that adolescent nicotine exposure slowed learning without affecting rats' cognitive strategy in the task. This data pattern suggests that exposure to nicotine in adolescence may have impaired different aspects of adult stimulus-response discrimination learning processes in males and females, but left abstract rule learning processes relatively spared in both sexes. These effects converge with other findings in the field and reinforce the concern that adolescent nicotine exposure poses an important threat to cognitive capacity in adulthood.

Concurrent Cognitive Processes in Rat Serial Pattern Learning: Item Memory, Serial Position, and Pattern Structure.

Three experiments examined the processes mediating rat serial pattern learning for rule-consistent versus rule-violating pattern elements ("violation elements"). In all three experiments, rats were trained to press retractable levers in a circular array in a specific sequence for brain stimulation reward (BSR). Experiment 1 examined the role of lever location (L) and element serial position (SP) cues in rats' ability to learn to anticipate a violation element positioned at the end of a 24-element serial pattern. Rats with L cues either alone or in combination with SP cues learned to anticipate the violation element, whereas those with SP cues alone did not. Rats in groups L and L+SP underwent a series of transfers designed to remove various cues that might have controlled their performance on the violation element. Results indicated that intra-chamber lever location cues mediated performance on the violation element whereas performance on rule-consistent elements within pattern chunks was mediated by an internal mnemonic representation that was insensitive to changes in lever location cues. Experiment 2 examined whether rats could learn to use SP cues alone to anticipate a violation element if it was positioned earlier in a serial pattern. Rats learned to anticipate the violation element based on SP cues alone when it was located in SP6 in a 24-element pattern, but not when it was in SP12. Experiment 3 examined whether or not rats spontaneously encode information about chunk length and the serial position of phrasing cues in serial patterns. Rats were trained to a high criterion on the serial pattern used in Experiment 1, then were challenged with three probe patterns that manipulated both chunk length and overall pattern length. Results indicated that rats spontaneously encoded information regarding the serial position of phrasing cues in relation to chunk length. Thus, rats appear to use at least three cognitive processes concurrently in serial pattern learning tasks, namely, item memory involving external discriminative cues, counting- or timing-like processes for encoding serial position, and rule abstraction for encoding an internal representation of pattern structure.

Blocking in rat serial pattern learning.

Both associative and rule-learning theories have been proposed to account for rat serial pattern learning, but individually they are unable to account for a variety of recent behavioral and psychobiological phenomena. The present study examined the role of rule learning versus discriminative learning in rat pattern learning using a classic associative phenomenon: blocking. Rats learned to press levers in an 8-lever circular array according to a rule-based serial pattern, 123-234-345-456-567-678-781-812, where digits indicate the correct lever in the array for each trial. Each pattern presentation contained a chunk with a final element violation, such as 454 instead of 456. Rats learned in a first phase that a noise signaled the violation chunk; then, a concurrent spatial cue was added in a second phase. A test with spatial cues alone showed that blocking occurred. The results suggest that associative learning mediated cuing of violation elements. Taken together with other behavioral and psychobiological evidence already reported in the literature implicating rule learning when rats learn this pattern in this paradigm, these results implicate multiple concurrent learning processes in rat serial pattern learning.

Determinants of phrasing effects in rat serial pattern learning.

Two experiments investigated how brief pauses introduced into serial patterns as phrasing cues would affect pattern learning in rats. In Experiment 1, a 24-element pattern consisted of eight 3-element chunks, whereas a 20-element pattern consisted of four 5-element chunks. In both patterns, 3.0-s temporal pauses placed at chunk boundaries (synchronous phrasing cues) facilitated learning compared to no phrasing. Cues "out of sync" with pattern structure (asynchronous phrasing cues) facilitated learning for the 24-element pattern and retarded learning for the 20-element pattern. Evidence suggested that in the latter case, 3.0-s pauses served as "blank" trials that induced rats to "skip" to the next serial position in sequence. In Experiment 2, shorter 0.5-s pauses served as phrasing cues in the 20-element pattern of Experiment 1. Synchronous short cues facilitated learning, whereas asynchronous phrasing cues had no effect. Furthermore, removal of synchronous cues produced deficits in performance on formerly cued trials, whereas removal of asynchronous cues had no effect. The results of Experiment 2 support the notion that in both experiments phrasing cues served as discriminative cues and indirectly suggest that rats are concurrently sensitive to pattern element cues, extra-sequence cues (such as phrasing cues), and to the relative timing of sequential events.