Acute alcohol intoxication paired with appetitive reinforcement: effects upon ethanol intake in infant rats

A recent study suggested that infant rats process alcohol odor and/or taste during acute ethanol intoxication probably due to ethanol elimination via respiration and salivation. The present set of experiments was meant to analyze the possibility that this orosensory processing may act as a conditioned stimulus when an appetitive reinforcer is paired with the state of intoxication. In the first experiment it was observed that intragastric administration of a mildly intoxicating ethanol dose (1.5 g/kg), paired during postabsorptive time intervals with oral infusion of sucrose, was sufficient to promote a significant preference to ethanol. In Experiment 2 different doses of ethanol were either paired or explicitly unpaired with sucrose administration. The result reported in Experiment 1 was replicated and it was observed that a higher dose (3.0 g/kg) unpaired with the reinforcer resulted in alcohol aversions in terms of alcohol consumption patterns. However, when the reinforcer was paired with this dose, the aversion was inhibited. Finally, in the third experiment results indicated that preexposure to alcohol odor eliminates sucrose-conditioned alcohol preferences. These results indicate that, in physiologically immature rats, alcohol preference can be regulated by prior associative experiences involving the state of intoxication and consequences internal and/or inherent to this state.     

Interactions between perinatal and neonatal associative learning defined by contiguous olfactory and tactile stimulation

Tactile stimulation of the neonate, as performed by the mother during and after delivery, has been described as an effective unconditioned stimulus during early ontogeny (Leon, 1987; Ronca & Alberts, 1994). The present experiments examined the interaction between perinatal and neonatal learning determined by the explicit association between alcohol odor and vigorous body stimulation of the perinatal organism. In Experiment 1, rat fetuses were exposed to either alcohol or saline 10 min prior to cesarean delivery. The alcohol administration procedure here employed was sufficient to provide sensory contamination of the amniotic fluid but avoid fetal alcohol intoxication. Pups in the two prenatal treatments later experienced the smell of alcohol, tactile stimulation, or both stimuli explicitly paired or unpaired. Other postnatal groups were composed of pups that had no explicit experience with either experimental stimulus. Pups subjected to alcohol odor in utero displayed more overall motor activity in response to that odor than saline controls. The increased motor responses were further potentiated in pups that experienced additional postnatal alcohol odor paired with tactile stimulation. In Experiment 2, pups were exposed to alcohol in the amniotic fluid 10 or 30 min prior to birth. As previously demonstrated the memory acquired in utero appears highly dependent upon contingency between exposure to this particular scent and delivery procedures. Pups in both prenatal treatment groups were then exposed to alcohol odor paired or unpaired with tactile stimulation. Some control animals received no further experience with either stimuli. Those pups exposed to alcohol odor paired with tactile stimulation both pre- and postnatally later showed maximum motor activity elicited by the odor of alcohol. The results support the notion of fetal associative learning comprising alcohol's chemosensory cues and behaviorally activating stimuli. Furthermore, the conditioned response under analysis is potentiated whenever neonates are reexposed to contingent presentations of the elements that defined the original associative memory.     

Early (preweanling) recognition of alcohol's orosensory cues resulting from acute ethanol intoxication

Three experiments were conducted in order to analyze the possibility that during acute alcohol intoxication, preweanling rats process ethanol's orosensory cues. Intragastric administration of an ethanol dose equivalent to 3.0 g/kg resulted in peak blood alcohol levels greater than 250 mg% (Experiment 1). Twenty-four hours after receiving this dose, 11-day-old pups expressed an ethanol odor aversion in a locational test. This aversion response was not observed when pups were previously administered an ethanol dose resulting in blood alcohol levels lower than 150 mg% (Experiment 2). Nevertheless when subjects received such a lower dose and 30 to 60 min after administration suffered nociceptive stimulation, alcohol aversions were also detected in terms of alcohol ingestion patterns and ethanol odor aversions (Experiment 3). These results appear to indicate that orosensory ethanol processing takes place during acute ethanol intoxication probably due to ethanol elimination via respiration, salivation, and/or hematogenic olfaction processes.     

Weanlings' transfer of conditioned ethanol aversion from olfaction to ingestion depends on the unconditioned stimulus

Weanling (21-day-old) rats were exposed to an alcohol odor paired with either an interoceptive (apomorphine-induced illness) or exteroceptive (footshock-induced distress) reinforcer. Twenty-four hours later, ethanol preferences were measured in a locational olfactory test (ethanol vs lemon odor) or an ingestion test (5.6% v/v ethanol vs 0.25% v/v citric acid solution). Weanling rats expressed substantial olfactory aversions, independent of the reinforcer employed in conditioning. During the drinking test, however, only rats that had experienced the ethanol odor paired with internal malaise showed a significant reduction in the intake of the ethanol solution when compared to unpaired controls. Furthermore, rats that had experienced the ethanol odor paired with external distress drank significantly more of the ethanol solution than their controls. These results provide further evidence that olfactory experiences with ethanol can lead to changes in ethanol ingestion, and indicate that the nature of the unconditioned stimulus is critical in establishing the ingestive effect.     

Differential ethanol olfactory experiences affect ethanol ingestion in preweanlings but not in older rats

Preweanling (21 days old) and adult (60-80 days old) rats were exposed to ethanol odor either paired with the early stages of apomorphine-induced toxicosis, paired with the recovery from toxicosis, or unpaired with the induction of distress. Twenty four hours later, ethanol preferences were measured in a spatial olfactory test (ethanol vs lemon odor) or a drinking test (5.6% v/v ethanol vs 0.25% w/v citric acid solutions). During the olfactory test both young and adult rats expressed substantial ethanol odor aversions when previously exposed to this odor paired with toxicosis. However, changes in ethanol intake became apparent only in preweanling subjects. Preweanlings which received the ethanol odor paired with illness drank significantly less of the ethanol solution relative to controls, while subjects experiencing the odor paired with recovery from distress significantly increased their consumption of the ethanol solution. These prior aversive and appetitive olfactory experiences had no effect upon ethanol intake in adult rats. These results implicate both an ontogenetic and a sensorial factor in the regulation of ethanol intake.     

Ontogenetic differences in the association of gustatory and tactile cues with lithium chloride and footshock

Using a double dissociation design with 15% sucrose solution and exposure to a heat lamp (34 degrees C) as CSs and 0.3 M LiCl and 1-mA footshock as USs, the present study found 15-day-old rats to show selective associations similar to those found in adults. Five-day-old pups, however, expressed similar sucrose aversions when this solution was paired with either LiCl or footshock, whereas they expressed heat aversions after heat-LiCl but not heat-shock pairings. Results are discussed with respect to previous data on selective associations in pups, current theories on selective associations, and ontogenetic differences in stimulus selection.     

Weanling and senescent rats process simultaneously presented odor and taste differently than young adults

Weanling, young-adult, and senescent Wistar albino rats had a novel odor/taste stimulus or a single taste stimulus either paired or explicitly unpaired with the unconditioned stimulus, lithium chloride. Animals were then given a saccharin vs water preference test. Standard preference scores indicated that the odor competed with taste for association with the US in young-adult rats but potentiated the conditioned aversion to taste in weanling and senescent rats. Results were interpreted in terms of age-related attentional differences which were hypothesized to account for infantile amnesia and for the memory dysfunction typically observed in senescent animals.     

Ethanol-mediated taste aversions and state-dependency in preweanling (16-day-old) rats

Requirements for conditioning of an ethanol-mediated taste aversion in 16-day-old rat pups were examined. Experiment 1 demonstrated that preweanling rats are capable of acquiring, in two trials, an aversion to a 15% sucrose solution when followed by intragastric intubation of a 1.2 g/kg dose of 17% v/v ethanol, but not when followed by a 0.4 g/kg dose. Comparison was with control animals given sucrose followed by an equivalent volume isocaloric Half and Half. When the 0.4 g/kg dose of ethanol preceded sucrose presentation by 30 min (Experiment 2), the aversion was learned, suggesting that the effective delay between the sucrose and the critical consequences of the ethanol had been too long with the former procedure. Expression of the sucrose aversion required, however, the reinstatement of the context of intoxication--state-dependent retention. Finally, the results of Experiment 3B indicated that, in addition to the association between the sucrose and the aversive consequences of alcohol intoxication, the orosensory cues resulting from alcohol's direct elimination, via such processes as respiration and salivation, became associated with the appetitive properties of the sucrose. This was evidenced by a conditioned increase in preference for ethanol odor. Possible age-related differences in the ability to associate stimuli with alcohol's unconditioned consequences, and in state dependency are discussed.     

Pavlovian heart rate and jaw movement conditioning in the rabbit: effects of medial prefrontal lesions

An experiment was conducted in which jaw movements (JM) and heart rate (HR) were concomitantly assessed in rabbits during simple Pavlovian conditioning. A 2-s 1200-Hz tone was the conditioned stimulus (CS) and an intraoral 1-cc pulse of 0.5 M sucrose-water solution was the unconditioned stimulus (US). Sham and medial prefrontal (mPFC)-lesioned animals received paired CS/US training with a 70- to 75-dB CS and were compared with sham- and mPFC-lesioned animals that received explicitly unpaired CS/US presentations. The percentages of JM CRs were significantly greater in the paired than the unpaired groups, but mPFC lesions had no effect on this measure. Conditioned HR decelerations occurred only in the paired groups and then only during the first session of training. Moreover, these CS-evoked cardiac decelerations were somewhat attenuated by the mPFC lesion. CS-evoked HR accelerations, which were significantly greater in unpaired than in paired animals, occurred during the four subsequent sessions. These results suggest that a CS-evoked cardioinhibitory process, mediated by the mPFC, is engendered by Pavlovian appetitive conditioning, as has been previously demonstrated for aversive conditioning. However, during JM conditioning these inhibitory changes are quickly replaced by tachycardia, possibly related to increased nonspecific somatomotor activity, since the tachycardia was somewhat greater in the unpaired animals.     

Interactions between conditioned and unconditioned flavor preferences

Five experiments investigated how rats' conditioned preferences or aversions for aqueous odors paired with sucrose or salt are affected by their unconditioned response to those tastes. Rats preferred an odor paired with 30% sucrose over an odor paired with 5% sucrose when both were presented in 5% sucrose, but they showed no preference or, if thirsty, showed the reverse preference, when the odors were presented in 30% sucrose. These changes in conditioned preference corresponded to changes in the rats' unconditioned preference for the accompanying sucrose solution. Rats' conditioned aversions for odors paired with salt showed a similar dependence on their reaction to the accompanying salt solution. The results were interpreted as showing that conditioned and unconditioned flavor preferences combine additively, as if mediated by the same sensory representation.     

Differential regional expression of brain-derived neurotrophic factor following olfactory fear learning

We examined brain-derived neurotrophic factor (BDNF) mRNA expression across the olfactory system following fear conditioning. Mice received 10 pairings of odor with footshock or equivalent unpaired odors and shocks. We found increased BDNF mRNA in animals receiving paired footshocks in the multiple regions examined including the posterior piriform cortex (PPC) and basolateral amygdala (BLA). This was in contrast to the unpaired and odor-alone treatments, where BDNF mRNA was increased in the olfactory bulb (OB) and anterior piriform cortex (APC) only, but not the higher olfactory areas. We propose that odor exposure increases expression of BDNF in the OB and APC while the PPC and BLA increase BDNF mRNA only when associative learning occurs.     

pCREB in the neonate rat olfactory bulb is selectively and transiently increased by odor preference-conditioned training

Early olfactory preference learning in rat pups occurs when novel odors are paired with tactile stimulation, for example stroking. cAMP-triggered phosphorylation of cAMP response element binding protein (pCREB) has been implicated as a mediator of learning and memory changes in various animals (Frank and Greenberg 1994). In the present study we investigate whether CREB is phosphorylated in response to conditioned olfactory training as might be predicted given the proposed role of the phosphorylated protein in learning. On postnatal day 6, pups were trained for 10 min using a standard conditioned olfactory learning paradigm in which a conditioned stimulus, Odor, was either used alone or paired with an unconditioned stimulus, Stroking (using a fine brush to stroke the pup). In some instances stroking only was used. The pups were sacrificed at 0, 10, 30, or 60 min after the training. Using Western blot analysis, we observed that the majority of olfactory bulbs in conditioned pups (Odor + Stroking) had a greater increase in pCREB activation at 10 min after training than pups given nonlearning training (Odor only or Stroking only). The phosphorylated protein levels were low at 0 min and at 60 min after training. This is in keeping with the slightly delayed and short-lived activation period for this protein. The localization of pCREB increases within the olfactory bulb as seen by immunocytochemistry. Naive pups were not exposed to odor or training. There was a significantly higher level of label in mitral cell nuclei within the dorsolateral quadrant of the bulb of pups undergoing odor-stroke pairing. No significant differences were observed among nonlearning groups (Naive, Odor only, or Stroking only) or among any training groups in the granule or periglomerular cells of the dorsolateral region. The localized changes in the nuclear protein are consistent with studies showing localized changes in the bulb in response to a learned familiar odor. The present study demonstrates that selective increases in pCREB occur as an early step following pairing procedures that normally lead to the development of long-term olfactory memories in rat pups. These results support the hypothesized link between pCREB and memory formation.     

Developmental flavor experience affects utilization of odor, not taste in toxiphobic conditioning

Feeding experiences were varied in developing rats and the effects upon flavor neophobia and lithium chloride-induced flavor aversions were observed. In Experiment 1, nursing experience of neonate rats was reduced by artificial feeding via intragastric cannula; the rats then were tested with apple juice paired with lithium chloride injection at weaning or maturity. Conditioned aversions were not affected, but neophobia to novel apple juice was attenuated in artificially-reared rats tested at maturity. In Experiment 2, rats received enriched feeding experience after weaning, which consisted of (a) obtaining many complex flavors, a few of which were paired with poisoning, effortlessly in the home cage, or (b) foraging for various foods on an elevated maze. No dramatic effects on neophobia or conditioned taste aversion for saccharin water were apparent. In Experiment 3, rats were given experience after weaning with vanilla-scented water either paired or unpaired with quinine water, and then tested with the odor of almond or that odor compounded with saccharin water for neophobia and lithium-induced aversions. Flavor-experienced rats exhibited more pronounced odor conditioning and more resistance to extinction of the odor aversion after both simple and compound conditioning. In contrast, saccharin taste aversions were relatively unchanged. Apparently, enriched feeding and drinking experience facilitates the utilization of odor more than taste cues.     

Multiunit changes in hippocampus and medial geniculate body in free-behaving rats during acquisition and retention of a conditioned response to a tone

Multiunit activity (MUA) was recorded chronically in the hippocampus (CA3) and the medial geniculate body (mMG) during habituation to a tone followed by conditioning (tone paired with footshock) or pseudoconditioning (tone/footshock unpaired) in rats previously trained in a lever-pressing for food task (VI 60). In the conditioned group pairing tones with footshocks rapidly induced an increase in the initial CS-evoked response in the mMG, followed by the emergence of a hippocampal response and a marked conditioned suppression of lever-pressing to the tone. In contrast, in the pseudoconditioned group, the stimulus induced only transient cellular changes in the hippocampus and in the mMG, while no behavioral suppression to the tone could be seen. Moreover, presentations of the CS 45 days later induced multiunit and behavioral responses in both structures, only in the conditioned group. These results are used for discussion of the role of learning-induced changes in the sensory structure (mMG) as compared with changes in an associative structure (hippocampus), during acquisition and retention of a conditioned response.     

Development switch in neural circuitry underlying odor-malaise learning

Fetal and infant rats can learn to avoid odors paired with illness before development of brain areas supporting this learning in adults, suggesting an alternate learning circuit. Here we begin to document the transition from the infant to adult neural circuit underlying odor-malaise avoidance learning using LiCl (0.3 M; 1% of body weight, ip) and a 30-min peppermint-odor exposure. Conditioning groups included: Paired odor-LiCl, Paired odor-LiCl-Nursing, LiCl, and odor-saline. Results showed that Paired LiCl-odor conditioning induced a learned odor aversion in postnatal day (PN) 7, 12, and 23 pups. Odor-LiCl Paired Nursing induced a learned odor preference in PN7 and PN12 pups but blocked learning in PN23 pups. 14C 2-deoxyglucose (2-DG) autoradiography indicated enhanced olfactory bulb activity in PN7 and PN12 pups with odor preference and avoidance learning. The odor aversion in weanling aged (PN23) pups resulted in enhanced amygdala activity in Paired odor-LiCl pups, but not if they were nursing. Thus, the neural circuit supporting malaise-induced aversions changes over development, indicating that similar infant and adult-learned behaviors may have distinct neural circuits.     

Associative morphine tolerance in the rat: examinations of compensatory responding and cross-tolerance with stress-induced analgesia

The results of three experiments designed to examine the processes subserving associative tolerance to morphine's analgesic effects, as assessed by the tail-flick test, are reported. The experiments indicated that associative tolerance in male Holtzman rats was not subserved by conditioned compensatory responses but was cross-tolerant with an apparently nonopioid form of stress-induced analgesia (SIA). Experiment 1 showed that rats tested for morphine analgesia in a distinctive context that had been paired previously with morphine injections (5 mg/kg) were more tolerant than animals that had had this context explicitly unpaired with a series of morphine injections or animals that were drug-naive. There was no evidence of a conditioned compensatory response of hyperalgesia in animals given a saline injection in the presence of environmental stimuli that had been previously paired with morphine, even under test conditions designed to minimize the possibility of floor effects that might have obscured the detection of drug-compensatory hyperalgesia. Experiment 2 demonstrated that the footshock procedures used for stress induction produced an SIA that was not attenuated by naloxone (10 mg/kg). Experiment 3 replicated the associative tolerance phenomenon of Experiment 1 and again failed to find evidence of conditioned compensatory responses. It was found that animals exposed to footshock in the context that had been associated with morphine administration developed significantly less SIA than control animals. The relevance of these findings for associative models of drug tolerance is discussed.     

The effects of prenatal stress on motivation in the rat pup

Exposure to prenatal stress (PNS) has been shown to induce a set of psychological and behavioral changes in developing offspring. We used the rodent model to investigate whether PNS produces changes in the ability of the pup to express social motivation. We used a set of behavioral tasks including monitoring ultrasonic vocalizations after isolation, a conditioned place preference, and a novel and familiar odor approach test. Pregnant Long-Evans rats were exposed to an unpredictable, variable stressor twice daily during the third week of gestation. Isolation vocalizations were assessed on postnatal day (PND) 10. Pup affinity for the dam was evaluated on PND 15. Typically, pups display a selective preference for an odor-paired environment only after the odor has been associated with the dam. This previous association produces a positive conditioned stimulus (CS). Normally, pups exposed to a neutral CS (odor paired with cotton balls) do not form this place preference. Results indicate that PNS exposed pups had significantly increased distress vocalizations and an equal preference for the positive and neutral conditioned stimuli. This type of alteration in forming early preferences could be detrimental because of decreases in the specificity of social learning and an impaired responsiveness in social relationships.     

Pavlovian conditioning with ethanol and lithium: effects on heart rate and taste aversion in rats

Rats received paired injections of either ethanol or saline as the conditioned stimulus and lithium chloride as the unconditioned stimulus (US) in a Pavlovian differential conditioning paradigm. Lithium chloride evoked a large deceleration in heart rate (80-100 beats per minute) as an unconditioned response. As a result of 10 conditioning trials, the substance paired with LiCl elicited a lower average heart rate than that elicited by the unpaired substance. Moreover, animals that received ethanol-LiCl injections subsequently were more averse to the taste of ethanol than animals receiving saline-LiCl pairings. However, there were no differences in ethanol's ability to serve as the US to induce an aversion to a novel flavor solution (i.e., the Avfail phenomenon was not observed). The overall pattern of results underscores the value of using multiple indexes of learning in drug-drug conditioning paradigms.     

Posttraining prefrontal lesions impair jaw movement conditioning performance,but have no effect on accompanying heart rate changes

This experiment examined the role of the medial prefrontal cortex (mPFC) in regulating learned autonomic and somatomotor responses in rabbits using appetitive Pavlovian conditioning. Interstimulus interval (ISI) duration [i.e., the time between the onset of the conditioned stimulus (CS) and unconditioned stimulus (US)] was manipulated in order to determine whether ISI duration was related to the heart rate (HR) responses obtained during conditioning. Two groups received either a 1- or a 4-s ISI, with a tone as the CS and an intraoral pulse of water as the US. Another two groups received explicitly unpaired presentations of either the 1- or 4-s tone CS and water US. Few conditioned jaw movement (JM) or HR conditioned responses (CRs) were observed in the unpaired conditions. Significant JM conditioning was, however, elicited by the paired conditions, especially to the 4-s ISI. Consistent CS-evoked HR accelerations were observed in both ISI conditions. After five sessions of training, the mPFC was lesioned in half the animals. A separate group of paired animals received sham lesions. After surgical recovery, all animals received 3 days of postoperative training. During the first postoperative training session, JM CRs significantly declined in both groups with mPFC lesions in comparison to the groups with sham lesions. The mPFC lesions, however, did not affect the CS-evoked cardiac accelerations, which again occurred during postoperative training.     

Alteration of Auditory Cortex Activity with a Visual Stimulus through Conditioning: A 2-Deoxyglucose Analysis

In two experiments, the 2-deoxyglucose metabolic mapping technique was used to examine the hypothesis that a stimulus of one modality (a light) will begin to activate the sensory cortex of a stimulus of another modality (a tone) with which it has been repeatedly paired. Adult gerbils received repeated presentations of either a light or the light paired with a tone known to affect 2DG labeling patterns in the auditory cortex. Intermittent footshock was included on a pseudo-random basis to maintain arousal in the subjects. One day after training, each gerbil was injected with 2DG and either received repeated presentations of the light only or was simply exposed to the training context. Analysis of the auditory cortex revealed no differences in overall metabolic activity of the auditory cortex between the groups. However, in both experiments, the light that was previously paired with the tone changed the relative activity of the cortical subfields compared to the light not previously paired with the tone. Specifically, the results indicate greater activity in the anterior auditory field (AAF—Experiments 1 and 2) and the posterior fields (DPVP—Experiment 2) relative to the primary field AI in response to the light that was previously paired with the tone during training. Gerbils either only placed in the context during the 2DG session or that received unpaired presentations of the light and tone during training did not show this shift in relative labeling between the subfields. Because no differences in overall activity of the auditory cortex were found, we conclude that the shift in relative labeling between the subfields reflects, on average, both an increase in activity of fields AAF and DPVP and a concomitant decrease in AI activity in response to the light stimulus. The results have implications for our understanding both of brain learning mechanisms in general and the potential functions of auditory cortex subfields in particular.