NMDA receptor blockade in the basolateral amygdala disrupts consolidation of stimulus-reward memory and extinction learning during reinstatement of cocaine-seeking in an animal model of relapse

Previous research from our laboratory has implicated the basolateral amygdala (BLA) complex in the acquisition and consolidation of cue-cocaine associations, as well as extinction learning, which may regulate the long-lasting control of conditioned stimuli (CS) over drug-seeking behavior. Given the well established role of NMDA glutamate receptor activation in other forms of amygdalar-based learning, we predicted that BLA-mediated drug-cue associative learning would be NMDA receptor dependent. To test this hypothesis, male Sprague-Dawley rats self-administered i.v. cocaine (0.6 mg/kg/infusion) in the absence of explicit CS pairings (2-h sessions, 5 days), followed by a single 1-h classical conditioning (CC) session, during which they received passive infusions of cocaine discretely paired with a light+tone stimulus complex. Following additional cocaine self-administration sessions in the absence of the CS (2-h sessions, 5 days) and extinction training sessions (no cocaine or CS presentation, 2-h sessions, 7 days), the ability of the CS to reinstate cocaine-seeking on three test days was assessed. Rats received bilateral intra-BLA infusions (0.5 microl/hemisphere) of vehicle or the selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate (AP-5), immediately prior to the CC session (acquisition), immediately following the CC session (consolidation), or immediately following reinstatement testing (consolidation of conditioned-cued extinction learning). AP-5 administered before or after CC attenuated subsequent CS-induced reinstatement, whereas AP-5 administered immediately following the first two reinstatement tests impaired the extinction of cocaine-seeking behavior. These results suggest that NMDA receptor-mediated mechanisms within the BLA play a crucial role in the consolidation of drug-CS associations into long-term memories that, in turn, drive cocaine-seeking during relapse.     

Cerebellar norepinephrine modulates learning of delay classical eyeblink conditioning: evidence for post-synaptic signaling via PKA

The neurotransmitter norepinephrine (NE) has been shown to modulate cerebellar-dependent learning and memory. Lesions of the nucleus locus coeruleus or systemic blockade of noradrenergic receptors has been shown to delay the acquisition of several cerebellar-dependent learning tasks. To date, no studies have shown a direct involvement of cerebellar noradrenergic activity nor localized the post-synaptic response to cerebellar beta-noradrenergic receptor signaling. Using ipsilateral, localized infusions into cerebellar lobule HVI and interpositus (IP), we have established that blocking beta-noradrenergic receptors with propranolol significantly impairs acquisition of conditioned responses. Furthermore, interrupting activation of cAMP-dependent PKA in the cerebellum using Rp-cAMPS completely prevents acquisition. However, neither blocking beta-adrenergic receptors nor blocking PKA activation significantly interferes with performance of established conditioned responses when administered after the learned response is formed.     

Timing at the Start of Associative Learning

Some theories of associative learning imply that time plays a fundamental role in the acquisition process. Consistent with these theories, this paper presents evidence that the time from the onset of a conditioned stimulus (CS) until presentation of the unconditioned stimulus (US) is learned very rapidly at the start of training. We report two autoshaping studies and a study on aversive conditioning in goldfish in which we examine timing at the start of conditioning. We also review data from a number of other conditioning preparations, including fear-potentiated startle, appetitive conditioning in rats, and eyeblink conditioning in rabbits, that report conditioned response (CR) timing early in training. Acquisition speed and the very first expressions of conditioned responding often show sensitivity to the time of US presentation. In instances where temporal control is slowly expressed, it is likely due to performance factors, not to slow learning about time. In fact, the learning about time may be a necessary condition for associative learning.     

Classical Delay Eyeblink Conditioning in in 4- and 5-Month-Old Human Infants

Abstract-Simple delay classical eyeblink conditioning, using a tone conditioned stimulus (CS) and airpuff unconditioned stimulus (US), was studied in cross-sectional samples of 4- and 5-month-old healthy, full-term infants. Infants received two identical training sessions, 1 week apart. At both ages, infants experiencing paired tones and air-puffs demonstrated successful conditioning over two sessions, relative to control subjects who had unpaired training. Conditioning was not evident, however, during the first session. Two additional groups of 5-month-olds received varied experiences during Session 1, either unpaired presentations of the CS and US or no stimulus exposure, fol-lowed by paired conditioning during Session 2. Results from these groups suggest that the higher level of conditioning observed following two sessions of paired conditioning was not the result of familiarity with the testing environment or the stimuli involved but, rather, the result of retention of associative learning not expressed during the first conditioning session.     

Mediated conditioning and retrospective revaluation with LiCl then flavour pairings

It is becoming accepted that the associative strength of a cue can change in its absence, despite this being difficult to explain using existing theories of Pavlovian conditioning. To investigate the influence of timing on learning about the representation of an absent cue, lithium chloride (LiCl) or a flavour previously paired with LiCl was presented in a distinctive context that had previously been paired with a neutral target flavour. The former treatment produced an aversion to the target flavour whether the LiCl was presented 10 min before, or immediately after, exposure to the context. However, presenting the flavour associate of LiCl created an aversion to the target flavour only if it had been presented 10 min after LiCl during initial training. This pattern of results cannot be explained in the terms of a simple timing account, and it is proposed that an explanation will require different associative rules operating in simultaneous and successive training schedules.     

Roles of hippocampal GABA(A) and muscarinic receptors in consolidation of context memory and context-shock association in contextual fear conditioning: a double dissociation study

Contextual fear conditioning involves forming a context representation and associating it to a shock, both of which involved the dorsal hippocampus (DH) according to our recent findings. This study tested further whether the two processes may rely on different neurotransmitter systems in the DH. Male Wistar rats with cannula implanted into the DH were subjected to a two-phase training paradigm of contextual fear conditioning to separate context learning from context-shock association in two consecutive days. Immediately after each training phase, different groups of rats received bilateral intra-DH infusion of the GABA(A) agonist muscimol, 5HT(1A) agonist 8-OH-DPAT, NMDA antagonist APV or muscarinic antagonist scopolamine at various doses. On the third day, freezing behavior was tested in the conditioning context. Results showed that intra-DH infusion of muscimol impaired conditioned freezing only if it was given after context learning. In contrast, scopolamine impaired conditioned freezing only if it was given after context-shock training. Posttraining infusion of 8-OH-DPAT or APV had no effect on conditioned freezing when the drug was given at either phase. These results showed double dissociation for the hippocampal GABAergic and cholinergic systems in memory consolidation of contextual fear conditioning: forming context memory required deactivation of the GABA(A) receptors, while forming context-shock memory involved activation of the muscarinic receptors.     

Consolidation and long-term retention of an implanted behavioral memory

Hypothesized circuitry enabling information storage can be tested by attempting to implant memory directly in the brain in the absence of normal experience. Previously, we found that tone paired with activation of the cholinergic nucleus basalis (NB) does induce behavioral memory that shares cardinal features with natural memory; it is associative, highly specific, rapidly formed, consolidates and shows intermediate retention. Here we determine if implanted memory also exhibits long-term consolidation and retention. Adult male rats were first tested for behavioral responses (disruption of ongoing respiration) to tones (1-15 kHz), yielding pre-training behavioral frequency generalization gradients. They next received 3 days of training with a conditioned stimulus (CS) tone (8.0 kHz, 70 dB, 2s) either paired (n=7) or unpaired (n=6) with moderate electrical stimulation of the nucleus basalis (～ 65 μA, 100 Hz, 0.2s, co-terminating with CS offset). Testing for long-term retention was performed by obtaining post-training behavioral frequency generalization gradients 24h and 2 weeks after training. At 24h post-training, the Paired group exhibited specific associative behavioral memory, manifested by larger responses to the CS frequency band than the Unpaired group. This memory was retained 2 weeks post-training. Moreover, 2 weeks later, the specificity and magnitude of memory had become greater, indicating that the implanted memory had undergone consolidation. Overall, the results demonstrate the validity of NB-implanted memory for understanding natural memory and that activation of the cholinergic nucleus basalis is sufficient to form natural associative memory.     

Background illumination effects upon in vitro conditioning in Hermissenda

In the marine snail Hermissenda, associative learning can be accomplished by paired presentations of light and vestibular stimulation. It is generally assumed that associative learning depends upon the intensity or salience of the conditioned or unconditioned stimulus (CS and US, respectively). Accordingly, during Hermissenda conditioning a stronger dark adaptation is expected to render the CS (the light) more salient and hence facilitate association. We studied the influence of background illumination level using an in vitro pairing procedure in Hermissenda. This procedure allows one to assess the effect of conditioning upon a single cell, the B photoreceptor, which is implicated in this learning process. After 15 min of adaptation to a dim background light, B photoreceptors maintained a basal rate of firing, while after adaptation to complete darkness, they stopped firing. Paired and unpaired groups received 10 training trials in either a completely dark or a dim light environment. Although a trial to trial cumulative increase in excitability was found in the paired group trained in darkness, only the paired group trained under dim background light showed a higher input resistance and cell excitability 10 min after training. These results suggest that the background dim illumination was not needed for the induction but played a role in the maintenance of the pairing effect. Possible mechanisms for such a modulatory effect are discussed.     

Impaired associative taste learning and abnormal brain activation in kinase-defective eEF2K mice

Memory consolidation is defined temporally based on pharmacological interventions such as inhibitors of mRNA translation (molecular consolidation) or post-acquisition deactivation of specific brain regions (systems level consolidation). However, the relationship between molecular and systems consolidation are poorly understood. Molecular consolidation mechanisms involved in translation initiation and elongation have previously been studied in the cortex using taste-learning paradigms. For example, the levels of phosphorylation of eukaryotic elongation factor 2 (eEF2) were found to be correlated with taste learning in the gustatory cortex (GC), minutes following learning. In order to isolate the role of the eEF2 phosphorylation state at Thr-56 in both molecular and system consolidation, we analyzed cortical-dependent taste learning in eEF2K (the only known kinase for eEF2) ki mice, which exhibit reduced levels of eEF2 phosphorylation but normal levels of eEF2 and eEF2K. These mice exhibit clear attenuation of cortical-dependent associative, but not of incidental, taste learning. In order to gain a better understanding of the underlying mechanisms, we compared brain activity as measured by MEMRI (manganese-enhanced magnetic resonance imaging) between eEF2K ki mice and WT mice during conditioned taste aversion (CTA) learning and observed clear differences between the two but saw no differences under basal conditions. Our results demonstrate that adequate levels of phosphorylation of eEF2 are essential for cortical-dependent associative learning and suggest that malfunction of memory processing at the systems level underlies this associative memory impairment.     

Training and testing determinants of short-term associative suppression of phototaxic behavior in Hermissenda

Hermissenda crassicornis shows both short- and long-term retention of conditioning following light-rotation pairings. Previous research has shown that prolonged training (50 trials per session, three consecutive daily sessions) produces a suppression of phototaxis lasting for days. This long-term retention reflects associative learning processes, with little or no contribution of nonassociative learning. In contrast, both associative learning and nonassociative behavioral modification contribute to short-term retention following a single session of five pairing trials. In this paper, we describe important associative and nonassociative determinants of short-term changes in phototaxis. In Experiment 1, animals received successive hourly tests for phototaxis in either a horizontal or a vertical orientation. Repeated testing resulted in decreased phototaxis which was especially pronounced for animals tested horizontally. Experiments 2-4 demonstrated that both repeated handling of animals and repeated periods of dark adaptation prior to each phototaxic test contributed to the development of phototaxic suppression with repeated testing. These nonassociative influences on phototaxis interacted with the gravitational orientation employed during behavioral testing, being most pronounced for testing in the horizontal orientation. An implication of these findings is that attempts to demonstrate short-term pairing-specific suppression will be most successful when nonassociative contributions are minimized (by testing animals vertically). Experiment 5 tested this prediction. We also tested the influence of training light intensity and the stimulus specificity of conditioned suppression. Animals received either five paired or five random presentations of light and rotation. Training light intensity was either moderate or bright. Following training, animals were tested for either suppression of phototaxis or suppression of negative geotaxis, using either a horizontal or a vertical testing orientation. Consistent with previous results, horizontally tested animals exhibited pronounced nonassociative suppression following training. The use of a bright training light also produced nonassociative suppression. However, when trained with a light of moderate intensity and tested vertically, Hermissenda showed associative suppression of phototaxis (significant paired--random difference) but not geotaxis (no paired--random difference). In a final experiment we observed that the longer the period of dark adaptation (prior to testing) the longer the phototaxic latency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Stimulus generalization of conditioned eyelid responses produced without cerebellar cortex: implications for plasticity in the cerebellar nuclei

In Pavlovian eyelid conditioning and adaptation of the vestibulo-ocular reflex, cerebellar cortex lesions fail to completely abolish previously acquired learning, indicating an additional site of plasticity in the deep cerebellar or vestibular nucleus. Three forms of plasticity are known to occur in the deep cerebellar nuclei: formation of new synapses, plasticity at existing synapses, and changes in intrinsic excitability. Only a cell-wide increase in excitability predicts that learning should generalize broadly from a training stimulus to other stimuli capable of supporting learning, whereas the alternatives predict that learning should be relatively specific to the training stimulus. Here we show that deep nucleus plasticity, as assessed by conditioned eyelid responses produced without input from the cerebellar cortex, is relatively specific to the training conditioned stimulus (CS). We trained rabbits to a tone or light CS with periorbital stimulation as the unconditioned stimulus (US), and pharmacologically disconnected the cerebellar cortex during a posttraining generalization test. The short-latency conditioned responses unmasked by this treatment showed strong decrement along the dimension of auditory frequency and did not generalize across stimulus modalities. These results cannot be explained solely by a cell-wide increase in the excitability of deep nucleus neurons, and imply that an input-specific mechanism in the deep cerebellar nucleus operates as well.     

The level of cholinergic nucleus basalis activation controls the specificity of auditory associative memory

Learning involves not only the establishment of memory per se, but also the specific details of its contents. In classical conditioning, the former concerns whether an association was learned while the latter discloses what was learned. The neural bases of associativity have been studied extensively while neural mechanisms of memory specificity have been neglected. Stimulation of the cholinergic nucleus basalis (NBs) paired with a preceding tone induces CS-specific associative memory. As different levels of acetylcholine may be released naturally during different learning situations, we asked whether the level of activation of the cholinergic neuromodulatory system can control the degree of detail that is encoded and retrieved. Adult male rats were tested pre- and post-training for behavioral responses (interruption of ongoing respiration) to tones of various frequencies (1-15 kHz, 70 dB, 2 s). Training consisted of 200 trials/day of tone (8.0 kHz, 70 dB, 2 s) either paired or unpaired with NBs (CS-NBs = 1.8 s) at moderate (65.7+/-9.0 microA, one day) or weak (46.7+/-12.1 microA, three training days) levels of stimulation, under conditions of controlled behavioral state (pre-trial stable respiration rate). Post-training (24 h) responses to tones revealed that moderate activation induced both associative and CS-specific behavioral memory, whereas weak activation produced associative memory lacking frequency specificity. The degree of memory specificity 24 h after training was positively correlated with the magnitude of CS-elicited increase in gamma activity within the EEG during training, but only in the moderate NBs group. Thus, a low level of acetylcholine released by the nucleus basalis during learning is sufficient to induce associativity whereas a higher level of release enables the storage of greater experiential detail. gamma waves, which are thought to reflect the coordinated activity of cortical cells, appear to index the encoding of CS detail. The findings demonstrate that the amount of detail in memory can be directly controlled by neural intervention.     

Contextual Pavlovian conditioning in the crab Chasmagnathus

In contextual conditioning, a complex pattern of information is processed to associate the characteristics of a particular place with incentive or aversive reinforcements. This type of learning has been widely studied in mammals, but studies of other taxa are scarce. The context-signal memory (CSM) paradigm of the crab Chasmagnathus has been extensively used as a model of learning and memory. Although initially interpreted as habituation, some characteristics of contextual conditioning have been described. However, no anticipatory response has been detected for animals exposed to the training context. Thus, CSM could be interpreted either as an associative habituation or as contextual conditioning that occurs without a context-evoked anticipatory response. Here, we describe a training protocol developed for contextual Pavlovian conditioning (CPC). For each training trial, the context (conditioned stimulus, CS) was discretely presented and finished together with the unconditioned stimulus (US). In agreement with the CSM paradigm, a robust freezing response was acquired during the 15 training trials, and clear retention was found when tested with the US presentation after short (2 and 4 h) and long (1–4 days) delays. This CPC memory showed forward but not simultaneous presentation conditioning and was context specific and protein synthesis dependent. Additionally, a weak CPC memory was enhanced during consolidation. One day after training, CPC was extinguished by repeated CS presentation, while one presentation induced a memory labilisation–reconsolidation process. Finally, we found an anticipatory conditioned response (CR) during the CS presentation for both short-term (4 h) and long-term memory (24 h). These findings support the conditioning nature of the new paradigm.     

The effects of beta-noradrenergic receptor blockade on acquisition of eyeblink conditioning in 3-month-old F344 rats

There is evidence that blocking beta-noradrenergic receptors will cause deficits in some forms of learning. We investigated the effects of systemic injections of 1, 5, and 10 mg/kg doses of propranolol on acquisition of delay eyeblink conditioning in 3-month-old Fischer 344 rats. We presented a 3-kHz, 90-dB tone as a conditioning stimulus and a 6 psi airpuff as our unconditioned stimulus to freely moving rats. We monitored eyelid activity using EMG signals. The treatment subjects were injected with either propranolol or saline 0.5 h prior to daily training sessions. Two groups of control subjects, one receiving injections of saline and one receiving injections of 5 mg/kg propranolol, received daily training sessions with unpaired and randomized presentation of the tone and airpuff. Each daily training session for the treatment groups consisted of 27 paired training trials and 3 conditioned stimulus-alone training trials. Rats injected with saline vehicle or with 1 mg/kg propranolol achieved a 60% or better learned response rate within two training sessions. Rats injected with 5 or 10 mg/kg propranolol never achieved a response rate significantly different from animals that received unpaired, random presentations of the tone and airpuff stimuli. These results agree with prior studies from our lab that have shown a dose-dependent effect of beta-noradrenergic receptor blockade on learning in rabbit eyeblink conditioning as well as in a runway, motor learning paradigm. We believe that the beta-noradrenergic system plays an important role in learning and memory in more than one cerebellar-dependent learning paradigm.     

The establishment of flavor-flavor associations using a sensory preconditioning training procedure

In Experiment 1, rats drank two distinct flavors in sequence during preconditioning; during training, the second of these flavors was paired with a toxin. During testing, there was an aversion to the flavor not directly paired with the toxin. In Experiment 2, the time interval between the two flavors (0, 3, 9, and 27 sec) in the preconditioning phase was varied; learning occurred only if the flavors were separated by 9 sec or less. Experiment 3, using a 60-sec interstimulus interval also did not reveal learning. These results reveal that the temporal gradient for flavor-flavor associative learning is similar to conventional audio-visual sensory preconditioning delay gradients and different from those obtained in flavor-toxicosis experiments. The results are discussed in terms of their critical implication for Revusky's concurrent interference theory of associative learning.     

The differential effects of emotional salience on direct associative and relational memory during a nap

Relational memories are formed from shared components between directly learned memory associations, flexibly linking learned information to better inform future judgments. Sleep has been found to facilitate both direct associative and relational memories. However, the impact of incorporating emotionally salient information into learned material and the interaction of emotional salience and sleep in facilitating both types of memory is unknown. Participants encoded two sets of picture pairs, with either emotionally negative or neutral objects paired with neutral faces. The same objects were present in both sets, paired with two different faces across the sets. Baseline memory for these directly paired associates was tested immediately after encoding, followed by either a 90-min nap opportunity or wakefulness. Five hours after learning, a surprise test assessed relational memory, the indirect association between two faces paired with the same object during encoding, followed by a retest of direct associative memory. Overall, negative information was remembered better than neutral for directly learned pairs. A nap facilitated both preservation of direct associative memories and formation of relational memories, compared to remaining awake. Interestingly, however, this sleep benefit was observed specifically for neutral directly paired associates, while both neutral and negative relational associations benefitted from a nap. Finally, REM sleep played opposing roles in neutral direct and relational associative memory formation, with more REM sleep leading to forgetting of direct associations but promoting relational associations, suggesting that, while not benefitting memory consolidation for directly learned details, REM sleep may foster the memory reorganization needed for relational memory.     

Central cannabinoid receptors modulate acquisition of eyeblink conditioning

Delay eyeblink conditioning is established by paired presentations of a conditioned stimulus (CS) such as a tone or light, and an unconditioned stimulus (US) that elicits the blink reflex. Conditioned stimulus information is projected from the basilar pontine nuclei to the cerebellar interpositus nucleus and cortex. The cerebellar cortex, particularly the molecular layer, contains a high density of cannabinoid receptors (CB1R). The CB1Rs are located on the axon terminals of parallel fibers, stellate cells, and basket cells where they inhibit neurotransmitter release. The present study examined the effects of a CB1R agonist WIN55,212-2 and antagonist SR141716A on the acquisition of delay eyeblink conditioning in rats. Rats were given subcutaneous administration of 1, 2, or 3 mg/kg of WIN55,212-2 or 1, 3, or 5 mg/kg of SR141716A before each day of acquisition training (10 sessions). Dose-dependent impairments in acquisition were found for WIN55,212-2 and SR141716A, with no effects on spontaneous or nonassociative blinking. However, the magnitude of impairment was greater for WIN55,212-2 than SR141716A. Dose-dependent impairments in conditioned blink response (CR) amplitude and timing were found with WIN55,212-2 but not with SR141716A. The findings support the hypothesis that CB1Rs in the cerebellar cortex play an important role in plasticity mechanisms underlying eyeblink conditioning.     

High Level of Footshock during Inhibitory Avoidance Training Prevents Amnesia Induced by Intranigral Injection of GABA Antagonists

Disruption of synaptic activity of a number of cerebral structures (e.g., neostriatum, amygdala, and thalamus) produces marked deficits in retention of instrumentally conditioned behaviors. When animals are given a relatively high number of training trials or high intensities of footshock during learning, however, such disruption is considerably less effective. Since there is a close anatomical and functional relationship between the neostriatum and the substantia nigra, it was of interest to determine whether enhanced training with a high level of footshock would prevent the reported amnesic state induced by injections of GABA antagonists into the latter structure. Rats were trained in a one-trial inhibitory task, using 0.2 or 0.4 mA, and then injected with microgram quantities of picrotoxin or bicuculline into the substantia nigra and posterior region of the zona incerta; retention was measured 24 h later. Only those groups that had been injected into the nigra and trained with 0.2 mA showed amnesia. These results support the hypotheses that (a) the normal activity of a set of structures is essential for the development of memory consolidation and (b) after an enhanced learning experience these structures may participate in memory consolidation, but are not necessary for the occurrence of this process.     

Amygdala modulation of memory consolidation: interaction with other brain systems

There is a strong consensus that the amygdala is involved in mediating influences of emotional arousal and stress on learning and memory. There is extensive evidence that the basolateral amygdala (BLA) is a critical locus of integration of neuromodulatory influences regulating the consolidation of several forms of memory. Many drug and stress hormone influences converge in activating the release of norepinephrine (NE) within the BLA. Evidence from studies using in vivo microdialysis and high-performance liquid chromatography indicates that increases in amygdala NE levels assessed following inhibitory avoidance training correlate highly with subsequent retention. Other evidence indicates that NE influences on memory consolidation require muscarinic cholinergic activation within the BLA provided by projections from the nucleus basalis magnocellularis (NB). Evidence from several experiments indicates that activation of the BLA plays an essential role in modulating memory consolidation processes involving other brain regions. These findings provide strong support for the hypothesis that the BLA plays a critical role in regulating the consolidation of lasting memories of significant experiences.     

Associative learning acquisition and retention depends on developmental stage in Lymnaea stagnalis

Associative learning dependent on visual and vestibular sensory neurons and the underlying cellular mechanisms have been well characterized in Hermissenda but not yet in Lymnaea. Three days of conditioning with paired presentations of a light flash (conditional stimulus: CS) and orbital rotation (unconditional stimulus: UCS) in intact Lymnaea stagnalis results in a whole-body withdrawal response (WBWR) to the CS. In the current study, we examined the optimal stimulus conditions for associative learning, including developmental stage, number of stimuli, interstimulus interval, and intertrial interval. Animals with a shell length longer than 18 mm (sexually mature) acquired and retained the associative memory, while younger ones having a shell length shorter than 15 mm acquired but did not retain the memory to the following day. For mature animals, 10 paired presentations of the CS and UCS presented every 2 min were sufficient for the induction of a WBWR to the CS. Furthermore, animals conditioned with the UCS presented simultaneously with the last 2 s of the CS also exhibited a significant WBWR in response to the CS. Blind animals did not acquire the associative memory, suggesting that ocular photoreceptors, and not dermal photoreceptors, detected the CS. These results show that maturity was key to retention of associative learning.