Acute alcohol intoxication paired with aversive reinforcement: ethanol odor as a conditioned reinforcer in rat pups

Previous research has suggested that infant rats process ethanol sensory properties during acute alcohol intoxication. The present study was designed in order to examine if alcohol odor could act as an aversive conditioned stimulus after the organism experiences the state of intoxication paired with nociceptive stimulation (footshock). In a first experiment 11-day-old pups received intragastric alcohol administration (1.5 g/kg). At different postabsorptive intervals footshock was presented (0-30, 30-60, 60-90, or 90-120 min). An explicitly unpaired control group which experienced footshock prior to the state of intoxication was also employed. All animals were subsequently tested in terms of alcohol intake and ethanol locational odor preferences. Both assessments indicated that pups which were exposed to the unconditioned peripheral stimulus 30-60 min after receiving ethanol expressed strong alcohol aversions. In a second experiment pups were exposed to footshock during this postabsorptive interval. Twenty four hours later, pups experienced ambient ethanol odor paired with soft or rough texture surfaces. Differential texture aversions were registered in experimental animals when compared with controls which suffered the state of intoxication explicitly unpaired with footshock, or unpaired presentations of ethanol odor and the tactile stimuli under consideration. These results appear to support the hypothesis concerning orosensory processing during an acute state of intoxication. Additionally it seems that the hedonic value of sensory attributes of this drug varies as a function of associative processes occurring during such a state.     

Association between chemosensory stimuli and cesarean delivery in rat fetuses: neonatal presentation of similar stimuli increases motor activity

Recent studies conducted in this laboratory indicated that prenatal chemosensory stimulation followed by cesarean delivery strongly affected postnatal responsiveness to odors derived from the administered substances. The present experiments were performed in order to examine if an associative process was responsible for such effects. In Experiment 1 rat fetuses during Gestational Day 21 were exposed to a tenuous alcohol solution or to a lemon-containing solution either 40 or 10 min prior to cesarean delivery. All subjects were subsequently tested in terms of changes in neonatal motor activity when confronted with the odor of alcohol or lemon. Rats experiencing prenatal cues 10 min prior to delivery exhibited higher and differential responsiveness to the smell of these cues when compared to those experiencing similar solutions 40 min prior to delivery. In Experiment 2 each fetus sequentially experienced both cues. Subsequent tests confirmed that the delay between prenatal sensory experience and birth induction was critical in terms of significantly affecting olfactory-mediated motor responses. The results suggest that consequences related with cesarean delivery act as an unconditioned stimulus capable of being associated with orosensory cues present in the amniotic fluid.     

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.     

An inexpensive and automated method for presenting olfactory or tactile stimuli to rats in a two-choice discrimination task

An inexpensive and automated method for presentation of olfactory or tactile stimuli in a two-choice task for rats was implemented with the use of a computer-controlled bidirectional motor. The motor rotated a disk that presented two stimuli of different texture for tactile discrimination, or different odor for olfactory discrimination. Because the solid olfactory stimuli were placed outside the chamber in metal pods with a mesh at front for odor sampling, "washout" of odors between trials was not necessary. To avoid differential auditory cues from motor rotation, the stimuli were arranged such that on each trial the motor always rotated exactly one quarter revolution (in 1 s), left or right, to present the next stimulus at trial start. To illustrate the use of the equipment, 2 rats were trained on tactile discrimination and 2 rats on olfactory discrimination. The rats sampled the stimulus on the disk through a port on the back wall by sniffing at it (olfactory) or touching it (tactile). The task was a go-left/go-right discrimination with the stimulus on the disk being discriminative for which lever provided reinforcement. The rats reached a stable level above 90% correct after 21 and 32 training sessions for tactile and olfactory discrimination, respectively. The article outlines how the equipment was constructed from low-cost components. Inputs from and outputs to the equipment were implemented through the parallel port of a personal computer without the use of a commercial interface board. The method of automated and low-cost presentation of olfactory or tactile stimuli should be of use for a variety of experimental situations such as matching-to-sample and cross-modal discrimination.     

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.     

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.     

Inhibition of nitric oxide synthase impairs early olfactory associative learning in newborn rats

The present experiments examined the role of nitric oxide ( NO) in early associative olfactory learning in rats. A preference for peppermint odor was induced by pairing peppermint odor with tactile stimulation in Wistar rat pups, in either a repetitive training paradigm or in a one-trial olfactory learning paradigm. In a first experiment we studied the effect of nitric oxide synthase (NOs) inhibition on early olfactory learning in a repetitive paradigm, by systemic daily injections of NG-nitro-l-arginine methyl ester (l-NAME, 50 mg/kg, i.p.). In order to exclude possible deleterous effects of repeated injections of l-NAME, we explored in a second experiment the effect of a single inhibitor injection in a one-trial olfactory learning paradigm. Inhibition of NOs was performed by either administration of l-NAME (50 mg/kg, i.p.), or 7-nitroindazole (7-NI, 30 mg/kg, i.p.), a more selective inhibitor of the neuronal NOs. We showed that both l-NAME and 7-NI impaired early olfactory associative learning when given before training but not before subsequent testing. Considering that NOs neurons are already widespread in the central nervous system (the olfactory bulb included) during the first postnatal week, the sites where NO inhibition may have acted to impair olfactory learning are discussed. The mechanisms of action of NO in relation with other neurotransmitters known to be necessary for olfactory conditioning in rat pups remain to be established. Impairment by NO synthesis inhibition of the acquisition during the first postnatal week of an olfactory conditioning, but not its recall, suggests a role for NO at synapses involved in that learning.     

Olfactory learning in the one-day old rat: reinforcing effects of isoproterenol

Within 24 h of their birth-induced norepinephrine surge, rat pups were tested for effects of a beta-receptor agonist, isoproterenol, on olfactory learning. Experiment 1 found no effect of isoproterenol on conditioning by pairing an odor (CS) with intraoral saccharin infusions. There was, however, unexpectedly strong responding in the unpaired control condition, which had the same contingency between the CS and isoproterenol as the paired condition. Experiment 2 found that pairings of odor and isoproterenol alone were sufficient for enhancing responding to the odor. Experiment 3 determined that isoproterenol had acted independently as a US for associative conditioning rather than facilitating nonassociative learning by mere exposure to the odor. These effects of isoproterenol as a US are consistent with the results of previous studies with older rats.     

Attention and suppression affect tactile perception in reach-to-grasp movements

Reaching with the hand is characterized by a decrease in sensitivity to tactile stimuli presented to the moving hand. Here, we investigated whether tactile suppression can be canceled by attentional orienting. In a first experiment, participants performed a dual-task involving a goal-directed movement paired with the speeded detection of a tactile pulse. The pulse was either delivered to the moving or stationary hand, during movement preparation, execution, or the post-movement phase. Furthermore, stimulation was delivered with equal probability to either hand, or with a higher probability to either the moving or resting hand. The results highlighted faster RTs under conditions of higher probability of stimulation delivery to both moving and resting hands, thus indicating an attentional effect. For the motor preparation period, RTs were faster only at the resting hand under conditions where tactile stimulation was more likely to be delivered there. In a second experiment, a non-speeded perceptual task was used as a secondary task and tactile discrimination thresholds were recorded. Tactile stimulation was delivered concomitantly at both index fingers either in the movement preparation period (both before and after the selection of the movement effector had taken place), in the motor execution period, or, in a control condition, in the time-window of motor execution, but the movement of the hand was restrained. In the preparation period, tactile thresholds were comparable for the two timings of stimulation delivery; i.e., before and after the selection of the movement effector had taken place. These results therefore suggest that shortly prior to, and during, the execution of goal-directed movements, a combined facilitatory and inhibitory influence acts on tactile perception.     

Prenatal experience and postnatal perceptual preferences: evidence for attentional-bias in bobwhite quail embryos (Colinus virginianus)

Previous studies have indicated that concurrent multimodal sensory stimulation can interfere with prenatal perceptual learning. This study further examined this issue by exposing 3 groups of bobwhite quail embryos (Colinus virginianus) to (a) no supplemental stimulation, (b) a bobwhite maternal call, or (c) a maternal call paired with a pulsating light in the period prior to hatching. Experiments differed in terms of the types of stimuli presented during postnatal preference tests. Embryos receiving no supplemental stimulation showed no preference between stimulus events in all testing conditions. Embryos receiving exposure to the unimodal maternal call preferred the familiar call over an unfamiliar call regardless of the presence or absence of pulsating light during testing. Embryos exposed to the call-light compound preferred the familiar call only when it was paired with the light during testing. These results suggest that concurrent multimodal stimulation does not interfere with prenatal perceptual learning by overwhelming the young organism's limited attentional capacities. Rather, multimodal stimulation biases what information is attended to during exposure and subsequent testing.     

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.     

Importance of tactile and olfactory cues to the inhibition of the grasshopper mouse's attack through toxicosis

This study examined the effect that toxicosis paired with the presence of a distinctive texture had on the inhibition of the grasshopper mouse's predatory attack. The first experiment measured the context in which tactile cues would be most effective by presenting prey with various combinations of added stimuli. The combination of distinctive tactile, olfactory, and gustatory cues produced the longest lasting inhibition. Because certain manipulations also had an unintended weak odor associated with them, the second experiment measured the importance of a weak or strong odor to inhibition of a mouse's attack. A combination of strong odor and distinctive texture paired with toxicosis inhibited an attack more effectively than a weak odor and a similar texture. In a third experiment, toxicosis was paired with an acraea moth caterpillar which has a highly distinctive texture. This produced the longest lasting inhibition of mouse's attack observed thus far. The ecological significance of a combination of a distinctive texture and odor to the inhibition of the grasshopper mouse's attack is discussed.     

The development of olfactory preferences for artificial odors briefly experienced by the precocial spiny mouse young

Groups of pups, aged 2, 4, 6, 12, 16, and 18 days of the precocial murid rodent spiny mouse (Acomys cahirinus), were each exposed for 1.5 h to one of the experimental odors, cinnamon or cumin, present in the surrounding environment. The odors were not contingent upon home-cage odors or any other reinforcers. This simple exposure produced a strong preference for the exposed odor in multichoice preference tests carried out 48 h after the exposure in pups from groups aged 2 through 16 days. Older pups exposed to cinnamon or cumin at Day 18 did not show a significant preference for the familiar odor. Both experimental odors used were found to be neutral for control experimentally naive pups within a tested age span. These results point at a stimulus familiarity, rather than associative learning in the development of olfactory preferences in young spiny mouse pups.     

Effects of the periodic administration of odor or vibration on a 3-hr. vigilance task

The present study examined performance during 3 hr. of simulated sentry duty with and without the intermittent administration of low-level sensory stimuli (odor or vibration). For odor and control conditions, target-detection latency increased steadily over the course of the 3-hr. session. Administration of a tactile stimulus reduced the increase in detection latency compared to that found in odor and control conditions. For all conditions, there were no significant differences in target-detection frequency, shot accuracy, or friend-foe discrimination. Across all conditions, restlessness (motor activity) increased significantly the first hour and remained elevated for the rest of the session. Subjective measures of workload (NASA-TLX) indicated that the 3-hr. task rated high on physical demand, mental demand, frustration, and overall workload. These findings suggest that the intermittent delivery of a clearly detectable tactile stimulus can reduce reaction-time decrements that occur as time on task increases.     

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.     

Neonatal tactile stimulation enhances spatial working memory, prefrontal long-term potentiation, and D1 receptor activation in adult rats

Environmental stimuli during neonatal periods play an important role in the development of cognitive function. In this study, we examined the long-term effects of neonatal tactile stimulation (TS) on spatial working memory (SWM) and related mechanisms. We also investigated whether TS-induced effects could be counteracted by repeated short periods of maternal separation (MS). Wistar rat pups submitted to TS were handled and marked transiently per day during postnatal days 2–9 or 10–17. TS/MS pups were stimulated in the same way as TS pups and then individually separated from their mother for 1 h/day. Their nontactile stimulated (NTS) siblings served as controls. In adulthood, TS and TS/MS rats showed better performance in two versions of the delayed alternation task and superior in vivo long-term potentiation of the hippocampo–prefrontal cortical pathway when compared with controls. Furthermore, there were more doses of A77636 (a selective dopamine D1 agonist) to significantly improve SWM performance in TS and TS/MS rats than in NTS rats, suggesting that activation of prefrontal D1 receptors in TS and TS/MS rats is more optimal for SWM function than in NTS rats. MS did not counteract TS-induced effects because no significant difference was found between TS/MS and TS animals. These data indicate that in early life, external tactile stimulation leads to long-term facilitative effects in SWM-related neural function.     

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.     

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.     

Automated assessment of conditioning parameters for context and cued fear in mice

A behavioral technique often used to evaluate the cognitive performance of rats and mice is the fear conditioning paradigm. During conditioned fear experiments, freezing responses shown by rodents after exposure to environmental stimuli previously paired to an aversive experience provide a behavioral index of the animal's associative abilities. The present study examined the ability of a computer-controlled automated Freeze Monitor system for recording immobility behavior in mice. The sensitivity of the automated procedure to detect group differences caused by the application of various training protocols was also evaluated. Statistical analyses revealed significant positive correlations between immobility scores obtained with the automated apparatus and hand-scored data collected by a continuous or a time-sampling method. Behavioral patterns recorded by the computerized system were very similar to those obtained by the hand-scoring methods adopted. In particular, during context testing, exposure to environmental stimuli previously paired with a mild foot shock (unconditioned stimulus [US]) evoked increased immobility behavior in mice conditioned with the US compared with levels of immobility displayed by mice previously confined to the same contextual stimuli without receiving the US. Moreover, although during conditioned stimulus (CS) testing, mice previously exposed to the US displayed high levels of immobility when confined to environmental cues much different from those paired with the US (contextual fear generalization), both hand-scored and automated results revealed the effect of CS–US pairing (increased immobility) only in mice trained to associate the two stimuli (paired group) but not in mice exposed to both CS and US separated by a 40-sec time interval (unpaired group) or in mice receiving only the US (US group) during conditioning sessions. Overall, the results show associative conditioning measured in an automated apparatus and highlight the utility of obtaining both latency as well as beam interruption parameters.     

A single serotonergic modulatory cell can mediate reinforcement in the withdrawal network of the terrestrial snail

A cluster of 40 serotonergic cells in the rostral part of pedal ganglia of the terrestrial snail Helix lucorum was shown previously to participate in the modulation of withdrawal behavior and to be necessary during the acquisition of aversive withdrawal conditioning in intact snails. Local extracellular stimulation of the serotonergic cells paired with a test stimulus elicited a pairing-specific increase (the difference between paired and explicitly unpaired sessions was significant, p <.01) of synaptic responses to test stimulation in the premotor interneurons involved in withdrawal. This result suggested participation of serotonergic cells in mediating the reinforcement in the withdrawal network. Intracellular stimulation of only one identified Pd4 cell from the pedal group of serotonergic neurons paired with a test stimulus also significantly increased (the difference between paired and explicitly unpaired sessions was significant, p <.05) synaptic responses to paired nerve stimulation in same premotor interneurons involved in withdrawal. Morphological investigation of a cluster of pedal serotonergic neurons showed that only the Pd4 cell had branches in the parietal ganglia neuropile where the synapses of premotor withdrawal interneurons and of presynaptic neurons are located. The data suggest that a single serotonergic cell can mediate the reinforcement in the withdrawal network of the terrestrial snail. Patterns of responses of the Pd4 cells to tactile and chemical stimuli conform to the suggestion.