Fos and glutamate AMPA receptor subunit coexpression associated with cue-elicited cocaine-seeking behavior in abstinent rats

Cocaine-associated cues acquire incentive motivational effects that manifest as craving in humans and cocaine-seeking behavior in rats. We have reported an increase in neuronal activation in rats, measured by Fos protein expression, in various limbic and cortical regions following exposure to cocaine-associated cues. This study examined whether the conditioned neuronal activation involves glutamate AMPA receptors by measuring coexpression of Fos and AMPA glutamate receptor subunits (GluR1, GluR2/3, or GluR4). Rats trained to self-administer cocaine subsequently underwent 22 days of abstinence, during which they were exposed daily to either the self-administration environment with presentations of the light/tone cues previously paired with cocaine infusions (Extinction group) or an alternate environment (No Extinction group). All rats were then tested for cocaine-seeking behavior (i.e. responses without cocaine reinforcement) and Fos and AMPA glutamate receptor subunits were measured postmortem using immunocytochemistry. The No Extinction group exhibited increases in cocaine-seeking behavior and Fos expression in limbic and cortical regions relative to the Extinction group. A large number of Fos immunoreactive cells coexpressed GluR1, GluR2/3, and GluR4, suggesting that an action of glutamate at AMPA receptors may in part drive cue-elicited Fos expression. Importantly, there was an increase in the percentage of cells colabeled with Fos and GluR1 in the anterior cingulate and nucleus accumbens shell and cells colabeled with Fos and GluR4 in the infralimbic cortex, suggesting that within these regions, a greater, and perhaps even different, population of AMPA receptor subunit-expressing neurons is activated in rats engaged in cocaine-seeking behavior.     

Dopamine D1 or D2 receptor antagonism within the basolateral amygdala differentially alters the acquisition of cocaine-cue associations necessary for cue-induced reinstatement of cocaine-seeking

The basolateral amygdala complex has been implicated in the formation and utilization of cocaine-cue associations in rat models of cue-induced reinstatement to cocaine-seeking behavior. We have previously demonstrated the importance of dopamine inputs to the basolateral amygdala complex in the reinstatement of cocaine-seeking behavior following chronic cocaine self-administration. Here we show that selective blockade of amygdalar dopamine D1 and D2 receptors during acquisition of cocaine-cue associations has distinctive effects on subsequent conditioned-cued cocaine-seeking behavior. Male, Sprague-Dawley rats were first trained to self-administer i.v. cocaine on a fixed ratio 1 schedule for 5 days. Subjects then received bilateral, intra-basolateral amygdala complex infusions of a dopamine D1 receptor antagonist (SCH23390, 0.25-2.0 microg/side; experiment 1), a dopamine D2 receptor antagonist (raclopride, 0.625-5.0 microg/side; experiment 2), or vehicle just prior to a single classical conditioning session, during which a light+tone cue was discretely paired with passive infusions of cocaine in the absence of lever responding. Following five additional days of cocaine self-administration and 7-10 days of extinction training, animals underwent multiple tests for cue-induced reinstatement. SCH23390 (2.0 microg/side), administered at the time of cocaine-cue association only, produced an attenuation of reinstatement to cue-induced cocaine-seeking behavior. In contrast, low doses of raclopride potentiated, while a higher dose of raclopride attenuated cue-induced reinstatement. These results demonstrate unique contributions of D1 vs. D2 receptors in mediating dopamine inputs within the basolateral amygdala complex during the formation of cocaine-stimulus associations that are critical for cue-induced reinstatement.     

The neural circuitry underlying reinstatement of heroin-seeking behavior in an animal model of relapse

Reinstatement of extinguished drug-seeking has been utilized in the study of the neural substrates of relapse to drugs of abuse, particularly cocaine. However, limited studies have examined the circuitry that drives the reinstatement of heroin-seeking behavior in the presence of conditioned cues, or by heroin itself. In order to test the hypothesis that the circuitry underlying reinstatement in heroin-experienced animals would show overlapping, yet distinct differences from cocaine-experienced animals, we used transient inhibition of several cortical, striatal, and limbic brain regions during reinstatement of heroin-seeking produced by heroin-paired cues, or by a single priming dose of heroin. Rats lever pressed for i.v. heroin discretely paired with a conditioned stimulus (CS) during daily 3-h sessions for a period of 2 weeks, followed by daily extinction of lever responding. Subsequent reinstatement of heroin-seeking was measured as lever responding in the absence of heroin reinforcement. The first set of reinstatement tests involved response-contingent CS presentations following bilateral intracranial infusion of either a combination of GABA receptor agonists (baclofen-muscimol, B/M) or vehicle (saline) into one of 13 different brain regions. The second set of reinstatement tests involved a single heroin injection (0.25 mg/kg, s.c.) following either B/M or vehicle infusions. Our results showed that vehicle-infused animals reinstated to both CS presentations and a priming injection of heroin, while B/M inactivation of several areas known to be important for the reinstatement of cocaine-seeking also attenuated heroin-seeking in response to CS presentations and/or a priming dose of heroin. However, as predicted, inactivation of areas previously shown to not affect cocaine-seeking significantly attenuated heroin-seeking, supporting the hypothesis that the circuitry underlying the reinstatement of heroin-seeking is more diffusely distributed than that for cocaine.     

Muscarinic receptor antagonism in the basolateral amygdala blocks acquisition of cocaine-stimulus association in a model of relapse to cocaine-seeking behavior in rats

Recent evidence has demonstrated a critical role for the basolateral amygdala complex in the reinstatement of extinguished drug-seeking behavior produced by drug-paired cues. In the current study, we utilized a model of the acquisition and expression of cocaine-stimulus associative pairing in order to study the role of cholinergic input to the basolateral amygdala in mediating conditioned-cued reinstatement. Male, Sprague-Dawley rats were first trained daily to self-administer i.v. cocaine on a fixed ratio 1 schedule of reinforcement. The muscarinic acetylcholine receptor antagonist, scopolamine, was directly infused into the basolateral amygdala prior to: a) classically conditioned pairing of a tone+light stimulus with cocaine infusions (acquisition), or b) testing of conditioned-cued reinstatement following a period of withdrawal from cocaine and extinction of cocaine-paired lever responding. Infusion of scopolamine just prior to the classical conditioning trial produced a dose-dependent disruption of cocaine-seeking behavior maintained by cocaine-paired cues during the reinstatement test. In contrast, infusion of scopolamine prior to the reinstatement test had no effect on conditioned-cued reinstatement of cocaine-seeking behavior.These results indicate a crucial role for cholinergic innervation of muscarinic acetylcholine receptors in the basolateral amygdala during the formation, but not the expression, of stimulus-reward associations that mediate cue-induced cocaine-seeking behavior.     

Environmental living conditions introduced during forced abstinence alter cocaine-seeking behavior and Fos protein expression

Environmental enrichment (EE) introduced during abstinence from cocaine self-administration is protective in reducing cue-elicited incentive motivation for cocaine in rats. This study examined neural activation associated with this protective effect of EE using Fos protein expression as a marker. Rats were trained to press a lever reinforced by cocaine (0.75 mg/kg/0.1 mL infusion) and light and tone cues across 15 consecutive days during which they were all housed in isolated conditions (IC). Rats were then assigned to either remain in IC, or to live in pair-housed conditions (PC) or EE for 30 days of forced abstinence from cocaine. Subsequently, cocaine-seeking behavior (lever presses without cocaine reinforcement) elicited by response-contingent cue presentations was assessed for 90 min, after which the rats' brains were immediately harvested for Fos protein immunohistochemistry. EE attenuated, whereas IC enhanced, cue-elicited cocaine-seeking behavior relative to PC. Also, within the prelimbic and orbitofrontal cortices and basolateral amygdala, IC enhanced, whereas EE reduced, Fos expression relative to PC. Furthermore, EE attenuated Fos expression in the infralimbic and anterior cingulate cortices, the nucleus accumbens (core and shell), bed nucleus of the stria terminalis, and ventral tegmental area, evident as a reduction relative to both PC and IC. In contrast, IC enhanced Fos expression in the dorsal caudate putamen, substantia nigra, and central amygdala, evident as an increase relative to both PC and EE. These results suggest that EE blunts neural activation throughout the mesocorticolimbic circuitry involved in cue-elicited incentive motivation for cocaine, whereas IC enhances activation primarily within the nigrostriatal dopamine pathway. These findings have important implications for understanding and treating drug-conditioned craving in humans.     

Associative and non-associative blinking in classically conditioned adult rats

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

Flavor preferences conditioned by intragastric glucose but not fructose or galactose in C57BL/6J mice

The present study determined if mice, like rats, differ in their flavor conditioning responses to intragastric (IG) infusions of three common monosaccharide sugars. In Experiment 1, C57BL/6J mice were trained to drink a flavored saccharin solution (the CS+) paired with intragastric (IG) self-infusions of 16% glucose, fructose or galactose and a different flavored solution (the CS-) paired with IG water infusions during 22 h/day training sessions. The glucose infusions increased CS+ intakes during training and produced a strong CS+ preference (~87%) in two-bottle choice tests. In contrast, the fructose and galactose infusions reduced CS training intakes and did not condition a CS+ preference. Experiment 2 determined if reducing fructose and galactose concentration would enhance conditioning. However, IG infusions of 8% sugar also failed to condition CS+ preferences. The robust conditioning response to IG glucose confirms results obtained with rats, but the indifference of mice to IG fructose and galactose contrasts with preference and avoidance responses observed in rats. The effectiveness of glucose to condition preferences suggests an important role for glucose-specific sensors rather than gut "sweet" taste receptors in the postoral modulation of carbohydrate appetite.     

Flavor preferences conditioned by intragastric nutrient infusions in food restricted and free-feeding rats

The role of deprivation state in flavor preference conditioning by nutrients was investigated in rats fitted with intragastric (IG) catheters. In different experiments, food restricted (FR) and food ad libitum (AL) groups were trained to drink one flavored solution (CS+) paired with IG infusions of maltodextrin, corn oil, or casein and another flavored solution (CS-) paired with IG water infusions. Training intakes of the CS solutions were limited to equate the exposure of the FR and AL groups. The IG nutrient infusions conditioned flavor preferences in FR and AL groups which, in three of four experiments, were of similar magnitude. Food restriction did, however, increase the overall intake of the CS+ solutions during testing. Rats trained with one CS+ while food restricted and a second CS+ while food unrestricted showed similar preferences for the two CS+ flavors. Prefeeding AL rats to satiety with chow prior to daily training sessions did not prevent them from developing a preference for a CS+ paired with IG maltodextrin. These findings indicate that the postoral actions of nutrients are reinforcing in food sated as well as hungry rats.     

Flavor preferences conditioned by high-fat versus high-carbohydrate diets vary as a function of session length.

Intragastric (i.g.) infusions of fat and carbohydrate condition flavor preferences in rats, but different results have been obtained in studies using pure and mixed nutrient infusions. This experiment compared the preference conditioning effects of mixed high-carbohydrate (HC) and high-fat (HF) diet infusions during short-term and long-term sessions. In Experiment 1 food-deprived rats were given one flavored saccharin solution (CS+HC) paired with i.g. infusions of an HC liquid diet, a second flavor (CS+HF) paired with HF diet infusions, and a third flavor (CS-) paired with i.g. water infusions during 30-min one-bottle training sessions. In subsequent two-bottle tests (30 min/day), the rats preferred both CS+s to the CS- and preferred the CS+HC to the CS+HF. In Experiment 2, the same rats were trained and tested with the CSs and paired infusions during 22 h/day sessions with chow available ad lib. Both CS+s were again preferred to the CS-, but now the CS+HF was preferred to the CS+HC. When given additional 30-min choice sessions in Experiment 3 the rats showed no reliable preference for the CS+HC versus CS+HF under food-deprived or ad lib conditions. In Experiment 4, the rats were given 22-h CS+HC versus CS+HF choice sessions every other day. They showed no reliable CS preference during the first 30 min of each session, but reliably preferred the CS+HF during the remaining 21.5 h. These findings indicate that previously reported differences in preferences conditioned by pure versus mixed nutrient infusions are due to training procedures (session length, deprivation state) rather than to the type of nutrient infusions per se. The rats displayed different CS+HF versus CS+HC preferences as a function of test duration even after being given both short- and long-term training. Thus, short-term choice tests do not always predict the long-term intakes and preferences for high-fat and high-carbohydrate foods.     

Conditioned flavor preference and aversion: role of the lateral hypothalamus

Food-restricted rats with ibotenic acid lesions of the lateral hypothalamus (LH) and sham controls were trained to associate flavored solutions with positive or negative postingestive consequences. The LH rats learned to prefer a flavor that was paired with concurrent intragastric infusions of maltodextrin. Unlike controls, the LH rats failed to learn a preference for a flavor paired with delayed maltodextrin infusions and showed an attenuated preference for a flavor paired with concurrent fat infusions. The LH rats did not differ from controls in learning to avoid flavors paired with concurrent or delayed infusions of lithium chloride. These data indicate that the LH is not essential for all types of flavor-postingestive consequence conditioning but is critical for learning to associate flavors with delayed nutrient feedback.     

Appetitive conditioning in neonatal rats: Conditioned orientation to a novel odor

These experiments document a form of early appetitive learning in rats obtained using classical conditioning procedures. Some of the special determinants of this conditioning are described, as well as ontogenetic changes in the effectiveness of training procedures. Learning was apparent when deprived 3- and 6-day-old rats oriented to and maintained contact with a novel and normally aversive odor after this odor had been paired with oral infusions of milk (Experiment I). The effectiveness of the conditioning procedures depended on the temperature at which pups were trained (Experiment IB). Moreover, the reinforcing properties of milk infusions depended on deprivation (Experiment IC). This conditioned change in responsiveness to odor was specific to the odor that had been paired with milk (Experiment II) and was retained for at least 24 hr (Experiment III).     

Cocaine-taking and cocaine-seeking behaviors in rats remain stable after systemic administration of GYKI 52466: a non-competitive AMPA receptor antagonist

Given the posited role of enhanced AMPA-mediated synaptic transmission in relapse to drug seeking, we investigated whether systemic administration of the AMPA receptor antagonist GYKI 52466 inhibits cocaine-taking and cocaine-seeking behavior in rats. Rats were trained to self-administer cocaine until stable self-administration was achieved. Effects of GYKI 52466 (1, 3, or 10 mg/kg, i.v.) on cocaine self-administration were assessed. Animals were allowed to re-establish stable cocaine self-administration and were then behaviorally extinguished from drug taking. The effects of GYKI 52466 (3, 10 mg/kg, i.v.) on cocaine-induced reinstatement of drug-seeking behavior were assessed. We found that GYKI 52466 failed to inhibit cocaine-taking and cocaine-seeking in both the self-administration and reinstatement paradigms. We suggest that although AMPA receptors may be involved in cocaine reward and addiction, the AMPA receptor antagonist GYKI 52466 has low therapeutic potential for cocaine addiction treatment.     

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

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

Flavor preferences conditioned by intragastric fructose and glucose: differences in reinforcement potency

Many prior conditioning studies indicate that fructose, unlike glucose, has minimal postingestive reinforcing effects. Using a new training procedure, food-restricted rats were trained in alternate 20-h/day sessions with one flavored solution (CS+F) paired with intragastric (IG) infusions of 16% fructose and another flavor (CS-) paired with IG water. In subsequent two-bottle tests they showed a robust (85%) preference for the CS+F over the CS-. A third flavor (CS+G) was then paired with IG 16% glucose, and it was strongly preferred to the CS+F. When retrained 30 min/day with new flavors paired with IG fructose, glucose, or water the rats learned only a CS+G preference. When training was extended to 20 h/day, a CS+F preference developed. New rats trained 20 h/day with two-bottle access to CS+F and CS- paired with IG fructose and water failed to acquire a CS+F preference. Other rats rapidly developed a strong preference when trained with concurrent access to CS+G and CS- paired with IG glucose and water. These data indicate that both fructose and glucose generate postingestive reinforcing signals, but that the fructose signals are weaker and/or delayed relative to those produced by glucose.     

CLASSICAL CONDITIONING OF THE GALVANIC SKIN RESPONSE IN IMMOBILIZED CATS

Ten cats were immobilized with Flaxedil and given either paired conditioning trials or unpaired presentations of the CS and UCS in a study of classical GSR conditioning. The paired group received 5 adaptation trials followed by 40 paired tone-shock acquisition trials and 10 extinction trials. A pseudo-conditioning control group received unpaired trials with tone and shock, to control for pseudoconditioning and sensitization. It was found that the group receiving paired conditioning trials responded at a significantly higher magnitude than did control Ss, thus demonstrating classical conditioning in this preparation. Electromyograms were recorded for all Ss during the experimental sessions to monitor skeletal movements. The level of immobilization was such that no movements were seen in response to the CS. Analysis of the baseline skin resistance data revealed no significant differences which could have contributed to group differences in GSR magnitudes. It is concluded that skeletal movement was not a necessary concomitant of classical conditioning of the GSR.     

Conditioned acceptance and preference but not altered taste reactivity responses to bitter and sour flavors paired with intragastric glucose infusion

Nutrient-conditioned flavors preferences are thought to involve an increase in flavor palatability (hedonic evaluation). Consistent with this view is the recent finding that a sweet flavor paired with intragastric glucose infusions elicited more hedonic taste reactivity (TR) responses than did an alternative sweet flavor paired with intragastric water. The generality of this finding was examined by conditioning preferences for inherently avoided nonsweet flavors. Rats were trained in 20 h/day and then 30 min/day sessions with a CS+ flavor (sour citric acid or bitter sucrose octaacetate) paired with intragastric 16% glucose infusion, and the opposite flavor (CS-) paired with intragastric water. Glucose conditioning increased the CS+ acceptance in one-bottle tests and produced a 95% CS+ preference in two-bottle sessions. Yet, TR responses to brief intraoral infusions of the two CS flavors did not differ, even after extensive testing. Subsequent choice tests revealed that a 1% fructose solution was preferred to the CS-, whereas the CS+ was preferred to 1% and 2% fructose and equally preferred to 4%, 8%, and 16% fructose. These results indicate that strong nutrient-conditioned flavor preferences are not always associated with increased flavor palatability as measured by TR tests. Therefore, nonhedonic processes, perhaps increased incentive salience, appear to mediate the enhanced preference and acceptance conditioned by postingestive nutrient actions.     

Area postrema lesions impair flavor-toxin aversion learning but not flavor-nutrient preference learning

Rats with lesions of the area postrema (APX) or sham lesions were trained to associate flavored solutions with positive or negative postingestive consequences. The APX rats were similar to controls in learning preferences for flavors paired with concurrent intragastric infusions of maltodextrin or corn oil and for a flavor paired with delayed maltodextrin infusions. In contrast, the APX rats displayed impaired aversion learning for flavors paired with toxic drug treatments (lithium chloride infusion or methylscopolamine injection). The aversion learning deficit ranged from mild to total, depending on training procedures. These findings confirm the important role of the area postrema in flavor-toxin learning but provide no evidence for its involvement in flavor-nutrient conditioning.     

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

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

Sub-region specific contribution of the ventral hippocampus to drug context-induced reinstatement of cocaine-seeking behavior in rats

The ventral hippocampus (VH) plays critical roles in cue-induced and cocaine-primed reinstatement of cocaine seeking [Rogers JL, See RE (2007) Neurobiol Learn Mem 87:688–692]. Subregions of the VH make distinct projections to elements of the brain relapse circuitry that mediate drug context-induced reinstatement. Thus, the VH may also critically contribute to this form of cocaine seeking in a subregion-specific manner. Accordingly, this study evaluated the hypothesis that functional inactivation of the ventral hippocampus proper (VHp)—but not of the dentate gyrus (DG)—impairs cocaine seeking elicited by re-exposure to a drug-paired environmental context. Rats were trained to lever press for un-signaled i.v. cocaine infusions (0.15 mg/infusion) in a distinct environmental context (cocaine-paired context) followed by extinction training in a distinctly different context (extinction context). Subsequently, cocaine-seeking behavior (i.e., non-reinforced active lever responding) was assessed in either the previously cocaine-paired context or the extinction context. Rats received bilateral microinfusions of the GABA agonist cocktail, baclofen+muscimol (BM: 1.0/.01 mM), or vehicle into the VHp, DG, or the posterior dorsal hippocampus (pDH; extra-VH control) immediately before each test session. Exposure to the previously cocaine-paired context, but not the extinction context, reinstated extinguished cocaine-seeking behavior following vehicle pretreatment. BM pretreatment administered into the VHp, but not the DG or pDH, significantly attenuated drug context-induced cocaine seeking. These results indicate that the VH contributes to drug context-induced cocaine seeking in a subregion-specific manner, with the functional integrity of the VHp being necessary for memory or motivational aspects of drug-paired environmental stimuli that sustain stimulus control over goal-directed behavior.     

Flavor preferences conditioned by post-oral infusion of monosodium glutamate in rats

Monosodium glutamate (MSG), the prototypical umami source, can enhance preference for associated flavors in humans and rodents. Although MSG flavor preference has been attributed to its taste, vagally-mediated post-oral detection has also been demonstrated. Recent studies showed that water-restricted rats acquired a preference for a flavor paired with intragastric (IG) infusion of 60 mM MSG in rats. The present study extends this work by comparing MSG-based flavor conditioning in water- and food-restricted rats and testing the persistence of flavor preferences. Rats with IG catheters drank flavored solutions paired with volume-matched infusions of 60 mM MSG or water in daily 30-min sessions. Two training/test cycles were conducted, each with eight one-bottle training sessions followed by two two-bottle preference tests without infusions. Food- and water-restricted groups displayed similar preferences for the MSG-paired flavor. When non-reinforced testing was continued after the second cycle, the food-restricted group sustained its preference across three 2-day tests, but water-restricted rats lost their preference. Other food-restricted rats learned to prefer a flavor paired with intraduodenal infusion, indicating that gastric stimulation by MSG is not required. A third experiment showed that adding 2 mM of the nucleotide inosine monophosphate to the IG infusion of MSG did not significantly enhance flavor conditioning. Because MSG-based flavor preferences can be obtained with infusions that bypass the stomach, the site for detecting MSG reinforcement may be intestinal.