some forms , such as declarative memory , are explicit memories of objects , places , or events .
others forms , such as non - declarative memory , are implicit , like habits , skills , or priming , and do not require conscious awareness ( gilbert et al .
, 2001 ; squire , 2004 ) . while this has been an established taxonomy of learning , the boundaries are often vague and difficult to demarcate .
for instance , in associative learning there is a fine - grained distinction between declarative associative learning , which is dependent on the conscious association of events , and non - declarative unconscious associative learning , which occurs without awareness of the link between the related meaningful events ( shanks , 1995 ) . in an associative learning task ,
when two stimuli are systematically presented in a temporal sequence , a new relationship between these two items is learned . in classical trace learning , a neutral conditioning stimulus ( cs ) precedes and , therefore , causes the subsequent prediction of an emotionally charged or noxious stimulus ( unconditioned stimulus , us ; figure 1 ) . during a differential eye - blink trace conditioning task ,
a specific tone ( cs+ ) warns of a puff of air to the eye , whereas another tone ( cs ) does not .
the presence of an anticipatory eye - blink response , which is a conditioned response ( cr ) to the cs+ tone , is highly correlated with participants verbal report of the relationship between the stimuli presented ; this is known as the contingency ( dawson and reardon , 1973 ; clark and squire , 1998 ) .
in contrast , during a delay conditioning task , for which there is no gap between the neutral stimulus ( cs ) and the puff of air to the eye , blink responses ( cr ) are elicited despite the lack of awareness of the contingency ( clark and squire , 1998 , 1999 ; figure 1 ) .
furthermore , trace , but not delay , conditioning is strongly influenced by an expectancy of the us ( clark et al . , 2001 ) .
differential trace ( upper graph ) and delay conditioning ( lower graph ) for the eye - blink task . in trace conditioning
there is a silent period between a tone and a puff of air to the eye .
the cs+ tone is presented for 250  ms , followed by a 500-ms silent gap , before a 100-ms puff of air to the eye ( aversive stimulus ) .
a second neutral stimulus tone , cs , is presented alone . in delay conditioning ,
the tone lasts for 850  ms , covering the silent gap and co - terminating with the puff of air .
consequently , delay conditioning has been considered a hallmark of non - declarative learning and is systematically used as an associative learning task in vertebrates and invertebrates ( lavond et al . , 1993 ) .
in contrast , human trace learning is dependent on conscious awareness of the contingency between stimuli ( christian and thompson , 2003 ) .
moreover it is this fact that has made human trace learning a potential  turing - test  of consciousness ( koch , 2004 ) .
though this  turing - test  potential has been influential in behavioral neuroscience , three arguments could pose a serious challenge to this notion : ( 1 ) trace conditioning can be learnt by almost every animal , even invertebrate sea slugs ( glanzman , 1995 ) ; ( 2 ) trace conditioning can be elicited using subliminal stimuli ( esteves et al . , 1994 ) ; and ( 3 ) clinically defined unconscious patients might learn trace conditioning ( bekinschtein et al .
it is in addressing these three arguments that we will characterize the theoretical boundaries of consciousness of learning .
the evolution of wings can be used as an analogy for the evolution of conditioning . during the evolution of animals on this planet , wings appeared at least three times from three different ancestors .
primitive wingless insects known as bristletails used long antennae - like filaments at the ends of their bodies to glide down to tree trunks from forest canopies ( yanoviak et al . , 2009 ) . the second appearance , around 150 million years ago , involved the development of wings on dinosaurs .
some dinosaurs started to evolve lighter skeletons ; their wrists changed and feathers grew to form wings ( lewin , 1983 ; sullivan et al . , 2010 ) . though dinosaurs evolved wings , which gave birth to modern birds their capacity to fly has a different origin and ancestry than flying insects .
the third appearance of wings developed in bats ( simmons et al . , 2008 ) .
though the three different wings serve a similar function , that is to fly , they were not derived from a common ancestor .
moreover , they are not controlled by the same machinery , and they do not obey the same rules .
is the same true of conditioning ? has a conditioning mechanism appeared only once in evolution ?
while the well preserved molecular machinery may suggest this , findings at the systems level are disparate and may suggest a different conclusion ( barco et al . ,
molecular mechanisms underlying acquisition and consolidation of memory are in fact preserved in most species . however
, learning relies on different network mechanisms in humans and sea slugs ( takehara et al . , 2003 ) .
the plasticity of arrays of neurons and the ubiquitous evolutionary pressure of associative learning makes it extremely difficult to disentangle whether conditioning emerged only once during the course of evolution .
it could be that conditioning emerged several times from several ancestral sources just as wings did .
we are only left with the certainty that associative learning can be instantiated in simply a few neurons , as in sea slugs , or in millions of neurons , as in mammals .
humans show the highest degree of behavioral flexibility among animals , probably through a flexible network that relies on a frontoparietal hub .
this flexibility , however , comes at a cost that results in slow , limited computational capacity .
trace conditioning does not seem to escape from this rule ( zylberberg et al . , 2010 ) .
arbitrary associations in humans are instantiated in this routing system that appears to be intrinsically related to consciousness ( zylberberg et al . , 2011 ) .
on the contrary , in the sea slug these temporary associations may rely on direct connections ( grol et al . ,
motor relations which bypass the central routing system ( grol et al . , 2006 ) .
trace conditioning in sea slugs , where consciousness is not required , does not necessarily imply that consciousness does not play a role in human trace conditioning .
alone , it does not imply more than , for example , a bacteria in need of oxygen breathing under water makes implications about human respiration .
the sea slug aplysia californica only needs a few neurons to perform trace conditioning , while rabbits require hippocampal , frontal cortex , and cerebellar networks ( christian and thompson , 2003 ) .
in contrast , rabbits seem to be more flexible than gastropods in their learning abilities , but not more than insects , i.e. , fruit flies , ( heisenberg et al . , 2001 ) . in humans , as in rodents , trace conditioning is dependent of the hippocampus ( james et al .
, 1987 ; moyer et al . , 1990 ; clark and squire , 1998 ) while delay conditioning can be elicited without hippocampi ( ivkovich and stanton , 2001 ; woodruff - pak and disterhoft , 2008 ) .
delay conditioning seems to rely primarily on a functional cerebellum ( mccormick and thompson , 1984 ; mauk and thompson , 1987 ; gerwig et al . , 2007 ) .
functional imaging studies have shown that the hippocampus is activated by both trace and delay conditioning , but it is significantly more activated by trace ( cheng et al . , 2008 ) .
these studies of neuroanatomy and the function of associative conditioning may indicate declarative memory s high dependence on the hippocampi and neocortex , as opposed to non - declarative memory s need for these structures , which is minimal ( eichenbaum and cohen , 2000 ) .
further evidence of a causal link between awareness of the contingency and trace conditioning comes from human and non - primate mammalian data . in humans
the variability in trace conditioning responses and learning seems to be linked to attention ( lovibond and shanks , 2002 ; carter et al . , 2003 ) , suggesting a strong modulation by central cognitive processes . in healthy volunteers , increasing attentional load parametrically
this elegant experiment used the classic n - back task to engage attentional resources and working memory .
the engagement of these dramatically decreases the anticipatory responses to the us in trace conditioning .
interestingly , the interference paradigm used in humans to decrease conditioning has been replicated using mice ( han et al . ,
2003 ) , demonstrating a similar functional frontotemporal network supporting successful trace conditioning in a non - primate mammalian species . in this study mice heard a tone and received a foot shock immediately after ( delay conditioning ) or , alternatively , the mice received a silent gap between tone and shock ( trace conditioning ) .
two additional groups of mice experienced two lights flashing as interference for the tone  shock conditions .
learning was impaired by light interference in trace but not in delay conditioning . in mice the light flashing acted as  cognitive  interference , just as working memory load might cause interference in trace conditioning with humans . in both populations , i.e. , in humans and mice ,
the fact that attention is a key component of conscious learning and also that trace conditioning is affected by awareness may indicate that trace conditioning is a type of conscious learning .
there is general agreement that exposure to a contingency between conditioned stimulus ( cs ) and us will create an associative process called conditioning ( dickinson , 1980 ; rescorla , 1988 ) .
the difference between human and non - human animal models is that the former can easily produce a verbal or motor report of the relationship between the cs and us .
if humans can form an internal representation of the contingency and verbalize it or make a voluntary response , then this behavior is taken as evidence of conscious awareness ( lovibond and shanks , 2002 ) .
what if the cs is masked , or more generally , not accessible to verbal reports ?
this question is central to current theoretical discussions of the role of conscious processing in trace conditioning .
the single - process model , asserts that a sole propositional learning process mediates expression cr and the expectancy of us ( full network mapping ; lovibond and shanks , 2002 ) .
the dual - process model , however , claims that these behavioral responses , both cr expression and us expectancy , are expressions of two independent learning processes ( partial network mapping ; perruchet , 1985 ; morris et al . , 1999 ;
some studies find evidence in support of the single - process model ( daum et al .
, 1991 , 1992 ; manns et al . , 2000a , b ; weike et al . , 2007 ) . while the series of experiments performed by perruchet , destrebecqz , cleeremans , and colleagues ( destrebecqz and cleeremans , 2001 ; destrebecqz et al .
, 2005 , 2010 ; perruchet et al . , 2006 ) and experiments performed by others ( ohman and soares , 1993 , 1994 , 1998 ; ohman et al . , 1995 ; weidemann et al . ,
the discussion is centered on the verbal reports of awareness of the contingencies ( in questionnaires , motor evaluation or subjective ratings ) and the measures of cr .
firstly , post - training questionnaires may elicit metacognitive process . therefore the participant may verbally report the contingencies , not because they noticed them during the learning phase , but because they were forced to think about the contingencies after conditioning had finished . in this case
it is difficult to know when conscious learning of the relationship between the stimuli occurred .
it could have been during learning or it could have been when that participant was prompted about the relationship between stimuli .
secondly , if awareness is measured online during the experiment , as to avoid a post hoc metacognitive process , then participants may then take note of the contingencies of which they were previously unaware .
the measurement of both verbal reports of contingencies and the cr is critical . in a series of studies , ohman et al .
( 1995 ) and ohman and soares ( 1993 , 1994 , 1998 ) argue that conditioning of electro - dermal responses to electric shocks can occur with masked , unconsciously perceived , stimuli .
it has been found that unconsciously perceived stimuli only elicited a cr when fear - relevant stimuli , such as spiders and snakes ( ohman and soares , 1993 ) or angry faces ( esteves et al . , 1994 ) , were presented as cs+ . in support of ohman
s claims , one recent study showed that sensitivity to masking conditions was related to the cr of a masked cs but not an unmasked cs ( cornwell et al . , 2007 ) . in this study ,
sensitivity to the masked condition was a marker of unconscious processing , i.e. , if participants were not aware of the masked item then this was taken to indicate that items were processed at an unconscious level .
the depth of unconscious processing of the cs was linked to the intensity of the cr .
weak perception of the cs through masking may not elicit conscious recognition of it , but the cs may still be above an identification threshold . however , there are criticisms of these findings .
one criticism is that the measure used to assess perceptual awareness of the cs may not be sufficiently sensitive to identify participants with residual awareness of stimulus features ( pessoa , 2005 ; graziano and sigman , 2009 ) .
additionally , some fear relevance effects in backward masking conditioning , as observed by ohman , could be due to selective sensitization rather than unconscious associative processes .
some methodological concerns have also been raised concerning the extent to which participants were truly unaware of the stimuli ( lovibond and shanks , 2002 ) .
firstly , not all verbal discriminative responses indicate awareness ; discrimination of stimuli above chance levels does not necessarily imply conscious awareness ( merikle and daneman , 2000 ; wiens and ohman , 2002 ) .
another highly debated experiment was performed by nez and de vicente ( 2004 ) , they have also showed that cr can be elicited when masked words are paired with a mild shock , and that this response is , as it was in the studies by ohman , related to the participants detection threshold .
however , the results of this study are somewhat difficult to interpret as a higher proportion of participants in the unconscious masked condition produced a cr than in the conscious group .
this study evaluated masked words paired with electric shocks ; they used either a detection threshold , i.e. , was the stimulus a word or a blank ? , or an identification threshold , i.e. , was it word1 , word2 or not a word ? when participants failed to detect a stimulus in the tachitoscope , half exhibited a cr above learning criterion ( four out of eight ) . yet , when participants were above detection threshold (  conscious  ) only 11% ( 2 of 18 ) showed learning .
there was a higher instance of conditioning , using a detection threshold , when stimuli were are presented unconsciously and when cr was measured using autonomic nervous system signals like skin conductance . on the contrary , when an identification threshold was applied , i.e. , subjects had to differentiate between two words or two non - words , only 10% ( 1 of 10 participants ) of participants in the unconscious condition exhibited cr , but 58% ( 7 out of 12 ) of participants in the conscious group exhibited cr .
these contradictory results point to two different learning systems , the unconscious system , which bypasses central processes and consciousness - related workspaces .
it also possibly directly links the early visual system with the autonomic nervous system and the conscious associative system , where the activation of the frontoparietal cortices may influence the autonomous nervous system giving rise to a different signal and a different type of learning .
it is indeed the case that the variability , speed , and regularity of the cr was higher in the conscious identification group as compared to the unconscious detection learners ( nez and de vicente , 2004 ) .
there are , however , caveats to this study s design that must be considered when discussing the conclusions .
firstly , in the identification threshold condition , words were repeated and could have therefore been deprived of their meaning .
when a word is repeated it becomes easy to rely on low level features , such as the letter array , to determine the word s identity without the need to access its meaning .
the participants decisions in this condition may not have been based on anything more than a surface features .
another methodological problem of this study is that the variability of the perceptual threshold was high and several subjects were therefore excluded leading to a small sample size .
the final subgroups of learners were 2/18 and 4/8 for conscious and unconscious detection of a word / blank , respectively , and 7/12 and 1/10 subjects for conscious and unconscious identification of the word , respectively .
the low number of participants complicates the statistical analysis and make conclusions difficult to extrapolate to other cases .
when an electric shock is used as the aversive stimulus , as opposed to a puff of air to the eye or a loud tone , it may induce a general increase in arousal . this may , change detection thresholds and act as a confounding factor , leading to difficulty in interpreting the results . in short
, there seems to be consistent evidence showing that trace conditioning can also be elicited by unconscious stimuli with a strong emotional content , which is not accessible for verbal report .
there may be some specific residual forms of trace learning that can be mediated by the most likely candidates : unconscious processing and emotional stimuli . however , evidence of subliminal abstract , non - emotional stimuli eliciting trace conditioning is not conclusive .
careful and well - designed experiments are needed to robustly deliminate the boundaries of consciousness thresholds of stimuli in a given task . their ability to elicit trace learning above and below this threshold should certainly be included in the agenda of highly relevant experiments in the next years .
if trace conditioning is taken as an indirect test of awareness , then a clinically defined unconscious patient , i.e. , those in a vegetative state ( vs ) , that shows crs ( learning ) should be considered conscious .
conversely , if a clinically defined unconscious patient shows anticipatory responses in trace conditioning , then this learning may not necessarily be indicative of conscious awareness ( bekinschtein et al . , 2009 ) .
they lack any behavior consistent with conscious awareness , such as , they do not follow someone with their eyes or head across the room , and they do not gesticulate or react to signs , words or commands . however , we used a classic differential trace conditioning eye - blink paradigm in 13 vs patients to investigate whether these unconscious patients might show learning ( bekinschtein et al . , 2009 ) .
moreover , we found a subset of these vs patients did show learning ( see figure 2 ) .
two vs could appropriately produce cr to cs+ and not to cs , and another four patients showed non - discriminatory anticipatory responses , i.e. , producing the cr to both stimuli types .
patients that demonstrated learning eventually recovered by regaining awareness , as opposed to those patients that did not show learning .
it is our belief that the patients that showed learning were partially conscious at the time of testing , but were unable to produce overt voluntary responses . in a recent trace conditioning study , using aversive noises and pleasant fanfares measuring skin conductance response , scott et al .
( 2011 ) , showed that only participants attending to the stimuli and able to catch the rule of the experiment showed crs .
if this paradigm is applied in the same disorders of consciousness patients group as the trace eye - blink conditioning , it may show convergence in conscious - dependent learning , and helping to better define the learning  consciousness relationship .
anticipatory learning in normal and anesthetized participants , and in vegetative state patients during trace differential conditioning .
bars show mean muscle activity for baseline of the cs+(bs cs+ ) , cs+ and baseline of the cs(bs cs ) , cs. learning is in arbitrary units .
sedated subjects show no learning ; vegetative state patients , as a group , show less muscle activity between cs+ and its baseline , and to cs. it is crucial to consider alternate hypotheses related to trace conditioning and the limited capacity of consciousness .
limited capacity implies that to learn trace conditioning , progressive gain of awareness of the contingency is required by maintaining a free global workspace clear of other contents ( dehaene and naccache , 2001 ) .
the use of this limited space for other events or processes may weaken the link that establishes the association between the cs and the us . this in turn would prevent the associative relationship from being established . in our original study
these sedated healthy participants showed no learning of the contingency , or cr , for eye - blink trace conditioning .
the sedative ( propofol ) that anesthetized these participants ensured that they were indeed unconscious , and the levels of drug caused a significant decrease in absolute cerebral blood flow .
in particular the propofol - related variations in the thalamic blood flow appeared to be linked to the midbrain reticular formation , thus suggests a close functional relationship between the two brain structures while unconscious ( fiset et al . , 1999 ) .
these deeply sedated participants showed true unconsciousness through both diminished awareness and arousal ( wakefulness ) .
moreover , the participants under low doses of propofol had a completely disorganized system for global integrated processing ( davis et al . , 2007 ; stamatakis et al . , 2010 ) . on the contrary ,
unconscious vs patients show dissociated wakefulness and awareness ; they are regarded as being awake but not aware ( jennett and plum , 1972 ) .
it could be that some vs patients retain partial functionality of the networks that support the acquisition of trace conditioning , but this is not enough to produce volitional movements .
sea slugs can learn trace conditioning , but they do not show a form of learning that reveals the flexibility typically displayed in conscious forms of learning ( van den bussche et al . , 2008 ; heinemann et al . , 2009 ; see dehaene and changeux , 2011 for a review ) .
instead , these mollusks use the minimal numbers of systems necessary for successful associative learning .
as far as we know sea slugs are not conscious , that is they are not conscious in the same way as humans are .
hence the mere observation of trace conditioning learning in this species does not provide evidence that trace conditioning is not an adequate signature of consciousness .
quite the opposite is true ; humans are extremely sensitive to context and are continuously interpreting all incoming stimuli in multiple ways .
this capacity for over - interpretation may help in social interaction , goal - oriented behavior and possibly changes the way that trace conditioning is encoded and processed .
the expression of trace learning in humans through a display of non - stereotyped representations of the contingency relies on an entirely distinct neural architecture
several studies have used masked or subliminally presented stimuli in order to make cs impossible to report ( unconsciously perceived ) .
this type of design has been used to explicitly determine whether conscious perception of the stimuli is necessary to achieve trace learning .
unfortunately , the results are inconclusive about whether trace learning , generally , is achievable through subliminal stimuli or under some specific circumstances .
when it is not neutral , but of negative valence , the masked stimuli seems to be processed up to the point of forming an association with the us , despite not being reported , detected , or discriminated .
the fact that clinically defined unconscious patients show trace learning suggest that they may have partial capacity for conscious awareness , as trace learning is dependant on some form of conscious awareness of the contingency .
this result is further strengthened by data from sedated participants that show drug induced unconsciousness does not produce anticipatory responses above the baseline ( bekinschtein et al . , 2009 ) .
trace conditioning remains very much linked to awareness of the contingency between cs and us .
we believe it is a combination of the timing between stimuli and the variations on the cs that will allow us to better frame , over the next few years , the boundaries between this basic form of learning and conscious awareness .
the aforementioned analyses leads us to propose here three conditions which should be met in order for trace conditioning to be used as a test for conscious awareness : ( 1 ) a relatively stable , sustained attention to the stimuli , ( 2 ) a low central processing load to avoid interference and ( 3 ) a well defined stimuli , perceptually discernible and close in presentation time .
if ( 1 ) is not met , then attention will deviate from the stimuli , and the creation of the association between cs and us will be disrupted .
this would lead to sparse and inconsistent demonstration of the cr ( armony and dolan , 2002 ) .
if ( 2 ) is not met then working memory capacity will be saturated by another task .
learning will then decrease , and as a result there will be low awareness of the contingencies . in this
respect , trace conditioning seems to require the central resources of the attentional system in order for the association to be established , and this seems to be paired with awareness ( carter et al . , 2003 ) .
if ( 3 ) is not met , then the stimuli will be ambiguous and the trace that is established will not be long - lasting or robust .
this will lead to partial learning only and high variance in cr ( lovibond and shanks , 2002 ; sehlmeyer et al . ,
we would like to raise one final important point that has not yet been emphasized .
it is of paramount importance if trace conditioning is to be used in practical terms .
clark and squire ( 1998 ) established that in order to observe an anticipatory eye - blink response participants must be aware of the contingency at a level that is sufficient for a verbal report .
the contrary , however , is not true ( both in the original clark and squire data and in our data making more than 200 participants combined ) : some participants show verbal reports of the contingency , but do not show the cr of an anticipatory associative response .
this simply may be because participants react in different ways to the anticipation of the air puff when they are aware that it is imminent .
the most frequent reaction is a contraction of the muscle prior to the air puff , however , some participants may control this spontaneous response by relaxing and , inhibiting their blinks or by simply doing nothing .
hence , an improved statement is needed . while the absence of learning does not provide information concerning the participants degree of awareness
the presence of trace learning may be taken as evidence in favor of conscious awareness .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 classical ( trace ) conditioning is a specific variant of associative learning in which a neutral stimulus leads to the subsequent prediction of an emotionally charged or noxious stimulus after a temporal gap . 
 when conditioning is concurrent with a distraction task , only participants who can report the relationship ( the contingency ) between stimuli explicitly show associative learning . 
 this suggests that consciousness is a prerequisite for trace conditioning . 
 we review and question three main controversies concerning this view . 
 firstly , virtually all animals , even invertebrate sea slugs , show this type of learning ; secondly , unconsciously perceived stimuli may elicit trace conditioning ; and thirdly , some vegetative state patients show trace learning . 
 we discuss and analyze these seemingly contradictory arguments to find the theoretical boundaries of consciousness in classical conditioning . 
 we conclude that trace conditioning remains one of the best measures to test conscious processing in the absence of explicit reports . 