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Alcohol-Mediated Taste Aversion in Infant Rats: Inhibitory effects of a stimulus paired with the onset of the state of intoxication

RM Pautassi, NE Spear & JC Molina.

Instituto Ferreyra, Argentina & Center Dev. Psychobiol., Binghamton Univ, USA.

Introduction

It was suggested that, at least in mice, ethanol would posses biphasic hedonic effects during the course of the toxic episode. Appetitive properties would be associated with the early stage of the toxic process, when blood alcohol’s concentracion (BAC’s) are rising; whereas aversive effects would be dominant in later moments associated with the highest BAC’s (Risinger & Cunningham, 1992). Differential conditioning during the same toxic process has also been reported with drugs such as morphine or amphetamine (Reicher & Holmann, 1977, Lett, 1998).

During early ontogeny of the rat, animals are not only capable to learn about chimiosensorial properties of ethanol (Molina & Chotro, 1989) but also regarding its unconditional properties (Molina et all, 1996; Abate et all, 2000).

Previous work from our laboratory (Pautassi et al, 2000) have indicated that, during rat’s early ontogeny, ethanol intoxication is able to support inhibitory as well excitatory aversive conditioned responses to cues presented during the course of the toxic episode. Specifically, it was shown that ethanol-induced saccharine aversions were completely inhibited if taste conditioning was evaluated under the presence of a tactile cue formerly paired with the onset of the state of intoxication. This effect suggest early ontogenetic processing of diferential unconditional properties of ethanol. Results are showed in figure I.

The aim of this experiment was to assess ethanol-induced taste aversion not only when tactile stimuli was simultaneously present but also when pups were exposed to this cue just before the flavor-conditioning evaluation. Magnitude of the conditioned response was also evaluated after reexposing the pups to the alcohol dose used during conditioning. Reexposition to some of the elements of the original learning situation defines a procedure known as rectivation treatment (Spear & Riccio, 1994). This procedure has been shown to facilitate the expression of infantile associative memories. In addition, was conducted a tactile preference location test compromising the ethanol-paired tactile cue and a novel one.  

Material and Methods

Conditioning: Experimental pups (paired animals) received an intragastrically administration of EtOH (2,5 g/kg) during posnatal days 14 (PD 14, conditioning day 1) and 15 (PD15, conditioning day 2). After administration, subjects were exposed to a tactile cue (sandpaper) during early stages of the alcohol-induced toxic process (5-15 min. post-administration). When blood alcohol reached peak levels (25-35 min. post-administration), the pups were intraorally stimulated with saccharine. Control subjects (unpaired animals) received both the drug and the exposition to tactile and sapid cues but explicity in a non paired schedule. With this arrangement of elements our goal was to generate an aversive excitatory response towards the sapid element, consequently procuring that the other conditioned stimulus (i.e. sandpaper), would enter into associative process with alternative effects of ethanol.

Testing: During PD 16, pups representatives of both conditioning treatments (paired or unpaired) were assigned to one of four groups. First group received a saccharine intake before a place location preference test (I-T group). Another group was evaluated regarding saccharine intake with the concurrently presence of the tactile cue sandpaper and then tested on place location preference (Simultaneous group: S). Two groups suffered reactivation treatments before being evaluated firstly on saccharine intake and finally in the place location preference test. One of those groups received as reactivation treatment a 10 minutes exposure to the tactile cue sandpaper [CS (I-T) group], while the other was administered with a 2,5 g/kg alcohol dose [US (I-T) group], four hours prior to evaluation. S groups were also evaluated regarding their locomotion while receiving saccharine infusions over sandpaper.

Saccharine infusions, both during conditioning and testing, were done using a standar peristaltic infusion pump (Manostat Casseste Pump). Tactile preference location test were performed by rooming the subjects into plexiglass opaques boxes. Half of the floor of this boxes was covered with the stimuli previously paired with the onset of the toxic process (i.e Sandpaper). The remaining half was covered with an alternative surface, the opposite side of a sandpaper unit. Animals could move on their own during ten minutes. Locomotion behavior was collected using an automatic software-based system (Ethovision, Holland). 

Results

With regards to test consumptions, a pertinent 2 X 4 ANOVA {Treatment: Paired or Non paired  X Order of Evaluation: I-T, [CS (I-T)], [US (I-T)] or S} revealed a main effect of treatment and type of evaluation [F(1,67)=26.05 p<0.0001, F(3,67)=5.103 p<0.005, respectively]. Interaction between factors  also reached significance [F(3,67)=4.005 p<.05]. Main factor effect and interaction were analyzed using Post hoc comparisons (Fisher’s LSD, p<.05). They revealed that ethanol-treated pups consume significantly less saccharine than control animals. The same result was seen in I-T groups, independently of treatment. When analysing the interaction it was clear that Ethanol I-T group consumed less saccharine than any other group, hence showing an aversion towards the sapid element. In contrast, no evidence of aversion was seen in Ethanol S and [CS (I-T)] groups. In short, they do not have differential consumption when compared with any of Non Paired groups. Ethanol [US (I-T)] group showed a moderate degree of aversion. This group consumed significantly more than pups from Ethanol I-T group but significantly less than those from Nonpaired groups. Results are presented in figure III.

Regarding Tactile preference locational test, data relative to percentage of time spent over sandpaper were analyzed with a 2 X 4 ANOVA (Treatment X Order of Evaluation). No evidence of a differential behavior was seen. No significant effect nor interaction was found.

No difference as a function of treatment was observed regarding locomotion of Simultaneous groups while receiving saccharine over sandpaper. Considering distance travelled, ANOVA (Treatment: Paired or Nonpaired X Interval of Evaluation: ten intervals of one minute each) only showed a significant effect of interval of evaluation [F(9,135)=5.18 p<0.0001]. With the help of post-hoc test (Fisher’s LSD) it was clear that pups moved less and less across the evaluation, expressing an habituation effect.  

Discusion

Tactile cue, which was paired with early stages of ethanol’s intoxication, seems to have aquired an specific memory which is opposite, or at least different, from the excitatory aversive one recruited by saccharine. Wheter the content of tactile memory is appetive or inhibitory is impossible to tell from previous results. An explanation in terms of a conditioned motor response elicited by sandpaper is dismissed as long as no differencial locomotor behavior was seen in Simultaneous Groups, independently of treatment.

In short, these results argue in favor of a hypothesis which suggest that ,early in life, preweanling pups are able to learn about differential effects of ethanol in terms of its competence to generate conditioned responses to exteroceptive cues.