Folliculin regulates mTORC1/2 and WNT pathways in early human pluripotency.

To reveal how cells exit human pluripotency, we designed a CRISPR-Cas9 screen exploiting the metabolic and epigenetic differences between naïve and primed pluripotent cells. We identify the tumor suppressor, Folliculin(FLCN) as a critical gene required for the exit from human pluripotency. Here we show that FLCN Knock-out (KO) hESCs maintain the naïve pluripotent state but cannot exit the state since the critical transcription factor TFE3 remains active in the nucleus. TFE3 targets up-regulated in FLCN KO exit assay are members of Wnt pathway and ESRRB. Treatment of FLCN KO hESC with a Wnt inhibitor, but not ESRRB/FLCN double mutant, rescues the cells, allowing the exit from the naïve state. Using co-immunoprecipitation and mass spectrometry analysis we identify unique FLCN binding partners. The interactions of FLCN with components of the mTOR pathway (mTORC1 and mTORC2) reveal a mechanism of FLCN function during exit from naïve pluripotency.

Systematic bias between running speed and metabolic power data in elite soccer players: influence of drill type.

The aims of the present study were to: i) evaluate the agreement between estimates of high-intensity activity during soccer small-sided games (SSGs) based on running speed alone and estimated metabolic power derived from a combination of running speed and acceleration; ii) evaluate whether any bias between the 2 approaches is dependent upon playing position or drill characteristics. 3 types of SSGs (5vs5, 7vs7 and 10vs10) were completed by 26 English Premier League outfield players. A total of 420 individual drill observations were collected over the in-season period using portable global positioning system technology. High-intensity activity was estimated using the total distance covered at speeds>14.4 km · h(-1) (TS) and the equivalent metabolic power threshold of > 20 W · kg(-1) (TP). We selected 0.2 as the minimally important standardised difference between methods. High-intensity demands were systematically higher (~100%, p<0.001) when expressed as TP vs. TS irrespective of playing position and SSG. The magnitude of this difference increased as the size of SSG decreased (p<0.01) with a difference of ~200% observed in the 5vs5 SSG. A greater difference between TP and TS was also evident in central defenders compared to other positions (p<0.05) particularly during the 5vs5 SSG (~350%). We conclude that the high-intensity demands of SSGs in elite soccer players are systematically underestimated by running speed alone particularly during "small" SSGs and especially for central defenders. Estimations of metabolic power provide a more valid estimation as to the true demands of SSGs.

A field-test battery for elite, young soccer players.

The validity and reliability of a battery of field-based performance tests was examined. The opinions of coaches, fitness professionals and players (n=170, 172 and 101 respectively) on the importance of performance testing were established using a questionnaire. On 2 occasions, separated by 7 days, 80 elite, young soccer players (mean±SD [and range]: age 13.2±2.6 [8.9-19.1] years; stature 1.59±0.18 m [1.32-1.91]; body mass 50.6±17.1 [26.5-88.7] kg) completed a battery of field-based tests comprised of heart rate response to a submaximal Multi-stage fitness test, 3 types of vertical jump, sprints over 10 and 20 m, and an agility test. Physical performance testing was considered important by coaches (97%), fitness professionals (94%) and players (83%). The systematic bias ratio and the random error components of the 95% ratio limits of agreement for the first and second tests, for the U9-U11 vs. U12-U14 vs. U15-U18 age groups, were [Systematic bias (*/÷ ratio limits)]: Heart rate (Level 5): 0.983 (*/÷ 1.044) vs. 0.969 (*/÷ 1.056) vs. 0.983 (*/÷ 1.055); Rocket jump: 0998 (*/÷ 1.112) vs. 0.999 (*/÷ 1.106) vs. 0.996 (*/÷ 1.093); 10 m sprint: 0.997 (*/÷ 1.038) vs. 0.994 (*/÷ 1.033) vs. 0.994 (*/÷ 1.038); Agility test: 1.010 (*/÷1.050) vs. 1.014 (*/÷1.050) vs. 1.002 (*/÷1.053). All tests, except heart rate recovery from the Multi-stage fitness test, were able to distinguish between different ability and age groups of players (p<0.05). Thus, the field-test battery demonstrated logical and construct validity, and was shown to be a reliable and objective tool for assessing elite, young soccer players.

The Football Association medical research programme: an audit of injuries in academy youth football.

OBJECTIVES: To undertake a prospective epidemiological study of the injuries sustained in English youth academy football over two competitive seasons. METHODS: Player injuries were annotated by medical staff at 38 English football club youth academies. A specific injury audit questionnaire was used together with a weekly return form that documented each club's current injury status. RESULTS: A total of 3805 injuries were reported over two complete seasons (June to May) with an average injury rate of 0.40 per player per season. The mean (SD) number of days absent for each injury was 21.9 (33.63), with an average of 2.31 (3.66) games missed per injury. The total amount of time absent through injury equated to about 6% of the player's development time. Players in the higher age groups (17-19 years) were more likely to receive an injury than those in the younger age groups (9-16 years). Injury incidence varied throughout the season, with training injuries peaking in January (p<0.05) and competition injuries peaking in October (p<0.05). Competition injuries accounted for 50.4% of the total, with 36% of these occurring in the last third of each half. Strains (31%) and sprains (20%) were the main injury types, predominantly affecting the lower limb, with a similar proportion of injuries affecting the thigh (19%), ankle (19%), and knee (18%). Growth related conditions, including Sever's disease and Osgood-Schlatter's disease, accounted for 5% of total injuries, peaking in the under 13 age group for Osgood-Schlatter's disease and the under 11 age group for Sever's disease. The rate of re-injury of exactly the same anatomical structure was 3%. CONCLUSIONS: Footballers are at high risk of injury and there is a need to investigate ways of reducing this risk. Injury incidence at academy level is approximately half that of the professional game. Academy players probably have much less exposure to injury than their full time counterparts. Areas that warrant further attention include the link between musculoskeletal development and the onset of youth related conditions such as Sever's disease and Osgood-Schlatter's disease, the significant number of non-contact injuries that occur in academy football, and the increased rates of injury during preseason training and after the mid season break. This study has highlighted the nature and severity of injuries that occur at academy level, and the third part of the audit process now needs to be undertaken: the implementation of strategies to reduce the number of injuries encountered at this level.

The Football Association Medical Research Programme: an audit of injuries in professional football--analysis of hamstring injuries.

OBJECTIVE: To conduct a detailed analysis of hamstring injuries sustained in English professional football over two competitive seasons. METHODS: Club medical staff at 91 professional football clubs annotated player injuries over two seasons. A specific injury audit questionnaire was used together with a weekly form that documented each clubs' current injury status. RESULTS: Completed injury records for the two competitive seasons were obtained from 87% and 76% of the participating clubs respectively. Hamstring strains accounted for 12% of the total injuries over the two seasons with nearly half (53%) involving the biceps femoris. An average of five hamstring strains per club per season was observed. A total of 13 116 days and 2029 matches were missed because of hamstring strains, giving an average of 90 days and 15 matches missed per club per season. In 57% of cases, the injury occurred during running. Hamstring strains were most often observed during matches (62%) with an increase at the end of each half (p<0.01). Groups of players sustaining higher than expected rates of hamstring injury were Premiership (p<0.01) and outfield players (p<0.01), players of black ethnic origin (p<0.05), and players in the older age groups (p<0.01). Only 5% of hamstring strains underwent some form of diagnostic investigation. The reinjury rate for hamstring injury was 12%. CONCLUSION: Hamstring strains are common in football. In trying to reduce the number of initial and recurrent hamstring strains in football, prevention of initial injury is paramount. If injury does occur, the importance of differential diagnosis followed by the management of all causes of posterior thigh pain is emphasised. Clinical reasoning with treatment based on best available evidence is recommended.

Rapid increase in clusters of presynaptic proteins at onset of long-lasting potentiation.

A change in the efficiency of synaptic communication between neurons is thought to underlie learning. Consistent with recent studies of such changes, we find that long-lasting potentiation of synaptic transmission between cultured hippocampal neurons is accompanied by an increase in the number of clusters of postsynaptic glutamate receptors containing the subunit GluR1. In addition, potentiation is accompanied by a rapid and long-lasting increase in the number of clusters of the presynaptic protein synaptophysin and the number of sites at which synaptophysin and GluR1 are colocalized. These results suggest that potentiation involves rapid coordinate changes in the distribution of proteins in the presynaptic neuron as well as the postsynaptic neuron.

The contribution of activity-dependent synaptic plasticity to classical conditioning in Aplysia.

Plasticity at central synapses has long been thought to be the most likely mechanism for learning and memory, but testing that idea experimentally has proven to be difficult. For this reason, we have developed a simplified preparation of the Aplysia siphon withdrawal reflex that allows one to examine behavioral learning and memory while simultaneously monitoring synaptic connections between individual identified neurons in the CNS. We previously found that monosynaptic connections from LE siphon sensory neurons to LFS siphon motor neurons make a substantial contribution to the reflex in the siphon withdrawal preparation (Antonov et al., 1999a). We have now used that preparation to assess the contribution of various cellular mechanisms to classical conditioning of the reflex with a siphon tap conditioned stimulus (CS) and tail shock unconditioned stimulus (US). We find that, compared with unpaired training, paired training with the CS and US produces greater enhancement of siphon withdrawal and evoked firing of LFS neurons, greater facilitation of the complex PSP elicited in an LFS neuron by the siphon tap, and greater facilitation of the monosynaptic PSP elicited by stimulation of a single LE neuron. Moreover, the enhanced facilitation of monosynaptic LE-LFS PSPs is greater for LE neurons that fire during the siphon tap and correlates significantly with the enhancement of siphon withdrawal and evoked firing of the LFS neurons. These results provide the most direct evidence to date that activity-dependent plasticity at specific central synapses contributes to behavioral conditioning and support the idea that synaptic plasticity is a mechanism of learning and memory more generally.

The association football medical research programme: an audit of injuries in professional football.

OBJECTIVES: To undertake a prospective epidemiological study of the injuries sustained in English professional football over two competitive seasons. METHODS: Player injuries were annotated by club medical staff at 91 professional football clubs. A specific injury audit questionnaire was used together with a weekly form that documented each club's current injury status. RESULTS: A total of 6,030 injuries were reported over the two seasons with an average of 1.3 injuries per player per season. The mean (SD) number of days absent for each injury was 24.2 (40.2), with 78% of the injuries leading to a minimum of one competitive match being missed. The injury incidence varied throughout the season, with training injuries peaking during July (p<0.05) and match injuries peaking during August (p<0.05). Competition injuries represented 63% of those reported, significantly (p<0.01) more of these injuries occurring towards the end of both halves. Strains (37%) and sprains (19%) were the major injury types, the lower extremity being the site of 87% of the injuries reported. Most injury mechanisms were classified as being non-contact (58%). Re-injuries accounted for 7% of all injuries, 66% of these being classified as either a strain or a sprain. The severity of re-injuries was greater than the initial injury (p<0.01). CONCLUSIONS: Professional football players are exposed to a high risk of injury and there is a need to investigate ways of reducing this risk. Areas that warrant attention include the training programmes implemented by clubs during various stages of the season, the factors contributing to the pattern of injuries during matches with respect to time, and the rehabilitation protocols employed by clubs.

Presynaptic role of cGMP-dependent protein kinase during long-lasting potentiation.

Previous research has suggested that cGMP-dependent protein kinases (cGKs) may play a role in long-term potentiation in hippocampus, but their site of action has been unknown. We examined this question at synapses between pairs of hippocampal neurons in dissociated cell culture. Injection of a specific peptide inhibitor of cGK into the presynaptic but not the postsynaptic neuron blocked long-lasting potentiation induced by tetanic stimulation of the presynaptic neuron. As controls, injection of a scrambled peptide or a peptide inhibitor of cAMP-dependent protein kinase into either neuron did not block potentiation. Conversely, injection of the alpha isozyme of cGK type I into the presynaptic but not the postsynaptic neuron produced activity-dependent potentiation that did not require NMDA receptor activation. Evidence from Western blots, reverse transcription-PCR, activity assays, and immunocytochemistry indicates that endogenous cGK type I is present in the neurons, including presynaptic terminals. These results support the idea that cGK plays an important presynaptic role during the induction of long-lasting potentiation in hippocampal neurons.

Parallel instabilities of long-term potentiation, place cells, and learning caused by decreased protein kinase A activity.

To further elucidate the links among synaptic plasticity, hippocampal place cells, and spatial memory, place cells were recorded from wild-type mice and transgenic "R(AB)" mice with reduced forebrain protein kinase A (PKA) activity after introduction into a novel environment. Place cells in both strains were similar during the first exposure and were equally stable for recording sessions separated by 1 hr. Place cell stability in wild-type mice was unchanged for sessions separated by 24 hr but was reduced in R(AB) mice over the longer interval. This stability pattern parallels both the reduced late-phase long-term potentiation in hippocampal slices from R(AB) mice and the amnesia for context fear conditioning seen in R(AB) mice 24 but not 1 hr after training. The similar time courses of synaptic, network, and behavioral instability suggest that the genetic reduction of PKA activity is responsible for the defects at each level and support the idea that hippocampal synaptic plasticity is important in spatial memory.

Is heterosynaptic modulation essential for stabilizing Hebbian plasticity and memory?

In 1894, Ramón y Cajal first proposed that memory is stored as an anatomical change in the strength of neuronal connections. For the following 60 years, little evidence was recruited in support of this idea. This situation changed in the middle of the twentieth century with the development of cellular techniques for the study of synaptic connections and the emergence of new formulations of synaptic plasticity that redefined Ramón y Cajal's idea, making it more suitable for testing. These formulations defined two categories of plasticity, referred to as homosynaptic or Hebbian activity-dependent, and heterosynaptic or modulatory input-dependent. Here we suggest that Hebbian mechanisms are used primarily for learning and for short-term memory but often cannot, by themselves, recruit the events required to maintain a long-term memory. In contrast, heterosynaptic plasticity commonly recruits long-term memory mechanisms that lead to transcription and to synpatic growth. When jointly recruited, homosynaptic mechanisms assure that learning is effectively established and heterosynaptic mechanisms ensure that memory is maintained.

Nitric oxide signaling contributes to late-phase LTP and CREB phosphorylation in the hippocampus.

Long-term potentiation (LTP) in the hippocampus has an early phase (E-LTP) that can be induced by one- or two-train tetanization, lasts approximately 1 hr, and is cAMP-dependent protein kinase (PKA) and protein synthesis independent and a late phase (L-LTP) that can be induced by three- or four-train tetanization, lasts >3 hr, and is reduced by inhibitors of PKA and of protein or RNA synthesis. Nitric oxide (NO) is thought to be involved in E-LTP, but until now there has been no information about the role of the NO-signaling pathway in L-LTP. We examined this question at the Schaffer collateral-CA1 synapses in slices of mouse hippocampus. An inhibitor of NO synthase blocked L-LTP induced by three-train tetanization and reduced L-LTP induced by four-train tetanization, whereas an inhibitor of PKA was more effective in blocking four-train L-LTP than three-train L-LTP. Three-train L-LTP was also blocked by inhibitors of guanylyl cyclase or cGMP-dependent protein kinase (PKG). Conversely, either NO or cGMP analogs paired with one-train tetanization produced late-phase potentiation, and the cGMP-induced potentiation was blocked by inhibitors of protein or RNA synthesis and an inhibitor of PKG, but not by an inhibitor of PKA. To test a possible downstream target of PKG, we examined changes in phospho-CRE-binding protein (phospho-CREB) immunofluorescence in the CA1 cell body area and obtained results similar to those of the electrophysiology experiments. These results suggest that NO contributes to L-LTP by stimulating guanylyl cyclase and cGMP-dependent protein kinase, which acts in parallel with PKA to increase phosphorylation of the transcription factor CREB.

The contribution of facilitation of monosynaptic PSPs to dishabituation and sensitization of the Aplysia siphon withdrawal reflex.

To examine the relationship between synaptic plasticity and learning and memory as directly as possible, we have developed a new simplified preparation for studying the siphon-withdrawal reflex of Aplysia in which it is relatively easy to record synaptic connections between individual identified neurons during simple forms of learning. We estimated that monosynaptic EPSPs from LE siphon sensory neurons to LFS siphon motor neurons mediate approximately one-third of the reflex response measured in this preparation, which corresponds to siphon flaring in the intact animal. To investigate cellular mechanisms contributing to dishabituation and sensitization, we recorded evoked firing of LFS neurons, the siphon withdrawal produced by stimulation of an LFS neuron, the complex PSP in an LFS neuron, and the monosynaptic PSP from an "on-field" or "off-field" LE neuron to an LFS neuron during behavioral training. Unlike the simplified gill-withdrawal preparation (Cohen et al., 1997; Frost et al., 1997), in the siphon-withdrawal preparation we found no qualitative differences between the major cellular mechanisms contributing to dishabituation and sensitization, suggesting that dissociations that have been observed previously may be attributable to transient inhibition that does not occur for this component of the reflex. Furthermore, in the siphon-withdrawal preparation, all of the various cellular measures, including monosynaptic PSPs from either on-field or off-field LE neurons, changed approximately in parallel with changes in the behavior. These results provide the most direct evidence so far available that both dishabituation and sensitization involve multiple mechanisms, including heterosynaptic facilitation of sensory neuron-motor neuron PSPs.

On the respective roles of nitric oxide and carbon monoxide in long-term potentiation in the hippocampus.

Perfusion of hippocampal slices with an inhibitor nitric oxide (NO) synthase blocked induction of long-term potentiation (LTP) produced by a one-train tetanus and significantly reduced LTP by a two-train tetanus, but only slightly reduced LTP by a four-train tetanus. Inhibitors of heme oxygenase, the synthetic enzyme for carbon monoxide (CO), significantly reduced LTP by either a two-train or four-train tetanus. These results suggest that NO and CO are both involved in LTP but may play somewhat different roles. One possibility is that NO serves a phasic, signaling role, whereas CO provides tonic, background stimulation. Another possibility is that NO and CO are phasically activated under somewhat different circumstances, perhaps involving different receptors and second messengers. Because NO is known to be activated by stimulation of NMDA receptors during tetanus, we investigated the possibility that CO might be activated by stimulation of metabotropic glutamate receptors (mGluRs). Consistent with this idea, long-lasting potentiation by the mGluR agonist tACPD was blocked by inhibitors of heme oxygenase but not NO synthase. Potentiation by tACPD was also blocked by inhibitors of soluble guanylyl cyclase (a target of both NO and CO) or cGMP-dependent protein kinase, and guanylyl cyclase was activated by tACPD in hippocampal slices. However, biochemical assays indicate that whereas heme oxygenase is constitutively active in hippocampus, it does not appear to be stimulated by either tetanus or tACPD. These results are most consistent with the possibility that constitutive (tonic) rather than stimulated (phasic) heme oxygenase activity is necessary for potentiation by tetanus or tACPD, and suggest that mGluR activation stimulates guanylyl cyclase phasically through some other pathway.

A prospective epidemiological study of injuries in four English professional football clubs.

OBJECTIVE: To define the causes of injuries to players in English professional football during competition and training. METHOD: Lost time injuries to professional and youth players were prospectively recorded by physiotherapists at four English League clubs over the period 1994 to 1997. Data recorded included information related to the injury, date and place of occurrence, type of activity, and extrinsic Playing factors. RESULTS: In all, 67% of all injuries occurred during competition. The overall injury frequency rate (IFR) was 8.5 injuries/1000 hours, with the IFR during competitions (27.7) being significantly (p < 0.01) higher than that during training (3.5). The IFRs for youth players were found to increase over the second half of the season, whereas they decreased for professional players. There were no significant differences in IFRs for professional and youth players during training. There were significantly (p < 0.01) injuries in competition in the 15 minute periods at the end of each half. Strains (41%), sprains (20%), and contusions (20%) represented the major types of injury. The thigh (23%), the ankle (17%), knee (14%), and lower leg (13%) represented the major locations of injury, with significantly (p < 0.01) more injuries to the dominant body side. Reinjury counted for 22% of all injuries. Only 12% of all injuries were caused by a breach of the rules of football, although player to player contact was involved in 41% of all injuries. CONCLUSIONS: The overall level of injury to professional footballers has been showed to be around 1000 times higher times higher than for industrial occupations generally regarded as high risk. The high level of muscle strains, in particular, indicates possible weakness in fitness training programmes and use of warming up and cooling down procedures by clubs and the need for benchmarking players' levels of fitness and performance. Increasing levels of injury to youth players as a season progresses emphasizes the importance of controlling the exposure of young players to high levels of competition.

Age-related defects in spatial memory are correlated with defects in the late phase of hippocampal long-term potentiation in vitro and are attenuated by drugs that enhance the cAMP signaling pathway.

To study the physiological and molecular mechanisms of age-related memory loss, we assessed spatial memory in C57BL/B6 mice from different age cohorts and then measured in vitro the late phase of hippocampal long-term potentiation (L-LTP). Most young mice acquired the spatial task, whereas only a minority of aged mice did. Aged mice not only made significantly more errors but also exhibited greater individual differences. Slices from the hippocampus of aged mice exhibited significantly reduced L-LTP, and this was significantly and negatively correlated with errors in memory. Because L-LTP depends on cAMP activation, we examined whether drugs that enhanced cAMP would attenuate the L-LTP and memory defects. Both dopamine D1/D5 receptor agonists, which are positively coupled to adenylyl cyclase, and a cAMP phosphodiesterase inhibitor ameliorated the physiological as well as the memory defects, consistent with the idea that a cAMP-protein kinase A-dependent signaling pathway is defective in age-related spatial memory loss.

An examination of the frequency and severity of injuries and incidents at three levels of professional football.

OBJECTIVE: To assess the risk of injury to professional footballers during European international and English Premier and First Division league matches. METHODS: Videotaped recordings of 29, 49, and 93 matches from the 1996 European Championship, 1996/1997 English Premier season and 1994 to 1997 English First Division seasons respectively were analysed. During each match, several relevant variables, including the number of fouls, injuries, time of incident, player identity, and injury mechanism, were recorded. RESULTS: Significantly more free kicks were awarded during international matches than during league matches; however, there were no significant differences between the numbers of free kicks awarded over the three First Division seasons assessed. Between 1.7 and 3.0% of fouls resulted in a player requiring treatment for injury, but only 15-28% of all injuries resulted from foul play. In all "non-foul" situations, in which injury resulted, at least 60% still involved player to player contact. No significant differences in injury frequency were observed between playing positions or match halves. CONCLUSIONS: The results equate to a total of 808 players per season from the estimated 2600 players in the four English professional football leagues sustaining a match injury that caused them to miss at least one game. The large number of underlying "non-injury" incidents is identified as the reason for this level of injury rather than a higher ratio of "injury" to "non-injury" incidents in professional football compared with other occupations.

Morphological changes associated with long-term potentiation.

Long-term potentiation (LTP) is a long-lasting form of synaptic plasticity induced by brief repetitive afferent stimulation that is thought to be associated with learning and memory. It is most commonly studied in the hippocampus where it may last for several weeks, and involves the synthesis of new proteins that might play a structural role. In this review we summarize the evidence in favor of modifications of neuronal architecture during LTP. We focus our attention on changes occurring at the level of single synapses, including components of postsynaptic dendrites (dendritic spines, the postsynaptic density, and synaptic curvature), of presynaptic terminals, and the formation of new synapses. We conclude that although many morphological changes at various sites have been observed during LTP, there is no definitive proof in favor of structural changes associated with LTP. However, morphological modifications remain a valid candidate for mechanisms of learning and memory.

Inducible and reversible gene expression with the rtTA system for the study of memory.

To obtain rapidly inducible and reversible expression of transgenes in the forebrain of the mouse, we have combined the reverse tetracycline-controlled transactivator (rtTA) system with the CaMKIIalpha promoter. We show that doxycycline induces maximal gene expression in neurons of the forebrain within 6 days and that this expression can be reversed by removal of doxycycline. Using calcineurin as a test transgene, we show that doxycycline-induced expression impairs both an intermediate form of LTP (I-LTP) in the hippocampus and the storage of spatial memory. The reversibility of the rtTA system in turn allowed us to examine the effects of the transgene on memory retrieval after normal storage had occurred. This examination suggests that retrieval requires some of the same molecular components required for storage.

Classical conditioning, differential conditioning, and second-order conditioning of the Aplysia gill-withdrawal reflex in a simplified mantle organ preparation.

Nonassociative learning was previously examined in a simplified preparation consisting of the isolated mantle organs and abdominal ganglion of Aplysia californica that is advantageous for relating cellular events to behavior (T. E. Cohen, S. W. Kaplan, E. R. Kandel, & R. D. Hawkins, 1997). Results of the current study show that the gill-withdrawal reflex in that preparation also underwent 2 associative forms of learning: classical conditioning and differential conditioning. In addition, the reflex underwent second-order conditioning with either forward or simultaneous pairing of a novel conditioned stimulus (CS2) and a previously conditioned stimulus (CS1). Moreover, extinction of CS1 after simultaneous second-order conditioning was accompanied by a decrease in responding to CS2, suggesting that the conditioning might have involved formation of an association between the CSs. In each of these paradigms, learning in the Aplysia mantle organ preparation resembled learning in vertebrates.