Operant conditioning with a stimulus discrimination: An alternative method for evaluating alcohol reinforcement in preweaning rats

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Highlight

  • Operant conditioning with stimulus discrimination in infant rats.

  • Alcohol self-administration evaluated by a stimulus discriminative procedure.

  • Alcohol reinforcement, infancy and operant discrimination of stimulus.

Abstract

Background

Ethanol exposure at early ontogeny promotes further predisposition to consume the drug. Operant conditioning allows motivational alcohol properties to be assessed. To date, the operant conditioning approach used during infancy consisted in paired subjects being trained to learn an operant response, using simultaneously a yoked partner, which received reinforcer solution as a result of a paired animal instrumental response (OYS).

New method

In our study, we attempted to evaluate ethanol reinforcing effects during PDs 15–18 in an operant conditioning schedule with a stimulus discrimination procedure (OSD), as an alternative control learning. This new proposal includes a single subject, who has to choose between an S+ nose-poke hole, which delivers the reinforcer into the mouth, or an S- nose-poke hole with no reinforcement effect.

Results

The OSD results seemed to be more reliable than those obtained using the OYS procedure, since some data appeared to be more robust when using a yoked nose-poke hole than when employing a yoked subject, such as in control learning. Consequently, OSD has the following advantages compared to the OYS procedure: a) the operant response learned is controlled by the overall behavior of the same subject, resulting in a relatively clearer data; b) a yoked animal is not necessary, thereby reducing the number of rats used in the operant conditioning procedure.

Comparison with existing methods and conclusions

A novel technique of operant conditioning adapted to infancy was developed by training animals to emit a particular response to gain access to alcohol solution as a reinforcer.

Introduction

Ethanol has rewarding effects, even in the early developmental stages. Experimental investigation, with rats or humans, has led to the proposal that ethanol exposure at early ontogeny acts as a challenge to promote a greater predisposition to consume the drug, compared with other ontogeny stages (Abate et al., 2008, Baer et al., 2003, Chotro et al., 2007, Spear and Molina, 2005).

Behavioral investigation carried out during infancy has some inherent complexity associated to developmental immaturity. For example, the behavioral repertory during infancy is restricted to breastfeeding, with a relatively low mobility (Branchi et al., 2001, Eilam and Golani, 1988, Geisler et al., 1993). Nevertheless, many diverse and alternative experimental approaches, usually employing rats, have been developed to study the impact of alcohol in early ontogeny, in terms of alcohol acceptability and voluntary consumption during this growth period. Infant rats consume all their necessary nutrients from breastmilk, so the voluntary intake of other solutions is not spontaneous. Thus, a classic procedure, which has been extensively applied, uses an alcohol intake test that consists in delivering ethanol via an infusion pump directly into the animal’s oral cavity through a cannula implanted in the cheek. This infantile intake test model consists of a semi-voluntary consumption task, by letting the pup drain the intake to avoid ingestion (Abate et al., 2001, Domínguez et al., 1998) During a later developmental period (early adolescence) in which feeding is totally independent from water consumption via a bottle –in the vivarium-, whole voluntary alcohol consumption can be assessed, and a two bottle choice test can be used to analyze alcohol preferences (Acevedo et al., 2010, Fabio et al., 2013). The infantile intake test is a useful technique for establishing that early ontogeny is a sensitive developmental period for studying alcohol reinforcement effects, characterized by a high acceptance and consumption of low alcohol concentrations (Abate et al., 2001, Domínguez et al., 1998).

The increasing interest in studying the motivational properties of alcohol in early ontogeny has led to the development of the artificial nipple technique (Varlinskaya et al., 1999). Rat neonates, rapidly attached themselves to an artificial nipple and had a voluntary intake of alcohol (Petrov et al., 2001; Varlinskaya et al., 1999). Furthermore, newborn rats pre-exposed to a nipple providing 5% or 10% v/v ethanol increased their subsequent responsiveness toward an empty surrogate nipple (no fluid) (Petrov et al., 2001). Overall, these results indicate that the artificial nipple was very useful not only for evaluating alcohol voluntary consumption, but also for assessing alcohol unconditioned properties. Although alcohol intoxication resulted in an increased response to an artificial nipple, this approach does not allow the possibility of discriminating between the positive reinforcement effects of the drug, or the calming effects of suckling behavior that counteract negative alcohol intoxication aspects (Abate et al., 2001). Also, age windows in which this technique could be employed are restricted to the first three days of rat life.

These antecedents discussed gave rise to a new alternative procedure for exploring motivational aspects of alcohol consumption, namely, operant conditioning. This technique specifically focuses on assessing the positive reinforcement of training animals regarding emitting a particular response (instrumental or operant response) to gain access to alcohol solution. Operant conditioning has been a widely used approach on adult rats, but the adaptation of this method to infant stages has been a challenge. Johanson and Hall (1979), described the first operant conditioning procedure for neonates, using milk as the reinforcer. This idea was then applied to evaluate alcohol reinforcing effects at PD 3, 9 and 15. An experimental subject (paired) was trained to pull a paddle resulting in milk with 6% of ethanol to be delivered directly to the oral cavity (Dominguez et al., 1993). Simultaneously, a second yoked control pup (a same-sex and age animal) was evaluated, with this subject receiving a similar oral infusion of the reinforcer each time the paired animal emitted the operant response. In other words, yoked controls and pair pups received equivalent reinforcement, but yoked control pups had no control over the contingency between target operant behavior and intraoral infusion. These results indicated that experimental pups rapidly learned to pull the paddle to self-administer milk contaminated with alcohol, compared with their yoked control (Dominguez et al., 1993).

Operant conditioning was used to assess alcohol reinforcement effects during early infancy, with specific adaptations incorporated in order to evaluate the particular responses of a pup’s behavioral repertory. Neonates usually display forelimb and head movements associated with stimulation of the mammary gland during breastfeeding. Based on this behavior, Bordner et al. (2008) adapted an experimental chamber to place neonates (PD1) in the supine position, thereby allowing free forelimb and head movements. A touch sensor was positioned in front of the pups, with forelimb or head contacts giving alcohol delivery into the oral cavity. Paired animals were evaluated together with a yoked subject. In this study, pups were trained to acquire an operant response to access an alternative alcohol solution (1.5% v/v, 3% v/v, 5%, 7.5% v/v or 10% v/v), which was followed by a second session –extinction- with no reinforcement contingency (Bordner et al., 2008).

The use of operant conditioning procedures with training and extinction phases to study ethanol self-administration allows the possibility of analyzing separately the consummatory (drinking) and appetitive (seeking) components of the response. During the training phase, it is possible to assess the amount of ethanol consumed and also some motivational effects of ethanol related to how hard subjects work to obtain access to ethanol. In addition, an extinction session allows the seeking response to be measured when the ‘expected’ ethanol delivery is over (Lopez and Becker, 2014).

An operant conditioning procedure was also adapted to 5-day old rats, with paired and yoked animals being evaluated in two daily training sessions during PDs 5 and 6. An extinction session took place immediately after the second training session was completed, with animals being placed in a semi-supine position with free forelimb and head movement (Arias et al., 2007). Reinforcers of 3% ethanol and 5% ethanol were used to assess the alcohol motivational effect in 5-day old rats, with the operant behavior to gain access to 3% ethanol being facilitated by prenatal exposure to ethanol during late gestation (Miranda-Morales et al., 2014).

During PDs 14–17, daily training sessions were performed and both subjects -experimental and yoked- were placed in an individual chamber with a hole in one of lateral walls, with a touch-sensitive sensor being located behind this hole. The target behavior under training was nose-poke. Specifically, each time the snout of an experimental subject touched the sensitive sensor, an infusion pump was activated which delivered a reinforcer solution into the oral cavity of both infant rats (Miranda-Morales et al., 2012a, Miranda-Morales et al., 2012b; Ponce et al., 2008).

To date, all the operant conditioning approaches used to evaluate alcohol motivational and consumption effects during infancy have included a particular learning control, with paired subjects being trained to learn an operant response and the consequence being a positive contingency of instrumental response and reinforcement. These animals were evaluated together with a yoked partner, which received the reinforcer solution as a result of a paired animal instrumental response (Arias et al., 2007, Bordner et al., 2008, March et al., 2009, Miranda-Morales et al., 2010, Miranda-Morales et al., 2012a, Miranda-Morales et al., 2012b, Miranda-Morales et al., 2014, Orellana Barrera et al., 2018, Orellana Barrera et al., 2017).

This study is the first approach to evaluate ethanol reinforcement effects in preweaning rats in an operant conditioning with a stimulus discrimination procedure (OSD) as alternative to an operant conditioning with a yoked subject (OYS). Typically, operant discrimination procedures (two lever discrimination) studies have been carried out in adult rats. Nevertheless, Johanson and Hall (1979) study trained 1-day-old rat pups in the presence of two paddles, discriminable on the basis of odor and position in the test container and the pups learned to probe into the paddle that provided them with milk. With these precedents and adapting operant conditioning procedure to evaluate infant rats, our proposal dispenses of the need for yoked animals, and learning control was performed by including a second hole with a sensitive touch -yoked nose-poke hole- in the conditioning chamber. Consequently, a single subject has to choose between the paired nose-poke hole (which delivered the reinforcer into the mouth) or the yoked nose-poke hole with no reinforcement effect.

Specifically, these experiments focused on two main goals. First, we aimed to analyze, with a conventional operant conditioning procedure –OYS-, the reinforcing attributes of ethanol in preweaning rats on PDs 15–18 that had no prior experience with the drug, replicating previous evidences. The second goal, an the most relevant for this work, was to examine the effectiveness of an alternative operant conditioning metrology –OSD-. Based on previous studies expected acquisition of the operant response to be obtained to gain access to alcohol as a reinforcer, under an OYS procedure, with similar results being expected using the OSD approach.

Prior studies on operant conditioning during infancy have included a particular control learning process, with the operant conditioning technique used during infancy including, in all cases, a paired subject involving experimental animals that were trained to learn the operant response. OYS procedure consists in the evaluation of an experimental subject with a simultaneous yoked condition. This control condition performed behavioral responses, but positive reinforcer being provided when the paired subject carried out the target behavior. As this control animal could not learn about contingency between operant response and reinforcement consequences, we ensured that the operant response of the paired animal was not a result of mere behavioral activation, but instead occurred due to a goal (Domjan, 2010, Miranda-Morales et al., 2012a, Miranda-Morales et al., 2012b, Orellana Barrera et al., 2017, Ponce et al., 2008). This OYS approach has been validated at different neonatal and infancy ages (Arias et al., 2007, Bordner et al., 2008, March et al., 2009, Miranda-Morales et al., 2010, Miranda-Morales et al., 2012a, Miranda-Morales et al., 2012b). In the present study, we attempted to evaluate alcohol reinforcing properties in an OSD, which consisted in an alternative control learning procedure.

Section snippets

Subjects

Wistar infant rats were employed in this study, born and reared in the INIMEC-CONICET-UNC vivarium. During the experimental period, the ambient conditions were controlled and the temperature was maintained at 21 °C with a 12 h dark cycle (light set at 8:00). Standard rat Purina chow and tap water were available ad libitum. Concerning reproduction, daily vaginal smears were obtained (between 10:00–12:00) to determine the stage of the estrous cycle of the female rats. Rats in proestrus were

%BWG during training session (PD15-17)

A 3-way mixed ANOVA was used, considering reinforcer solution (chocolate, 1.5% or 3.75% ethanol) as the between-subject factor, and days of training (PD 15, 16 or 17) and learning condition (paired or yoked-subjects) as the within-subject factors. However, this analysis failed to indicate significant effects, suggesting that this procedure would not be useful for evaluating differences in reinforcer consumption scores. This result indicates that paired and yoked animals are exposed at a similar

Discussion

The present study used operant conditioning to evaluate alcohol reinforcement during infancy, following a similar procedure previously reported in the literature (Miranda-Morales et al., 2012a, Miranda-Morales et al., 2012b, Ponce et al., 2008). The operant conditioning with a yoked subject (OYS) included a group of animals to control learning establishment. In this procedure, an experimental animal was trained to learn a nose-poke operant response simultaneously with a yoked pup, a learning

Author Note

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET PIP 11220120100223, P.A.), Agencia Nacional de Promoción Científica y Técnica (ANPCyT-FONCyT, PICT 0130, P.A.), Secretaría de Ciencia y Tecnología of the Universidad Nacional de Universidad Nacional de Córdoba (SECyT-UNC 2016-2017, P.A.) and fellowships from Secretaría de Ciencia y Tecnología Universidad Nacional of the Universidad Nacional de Córdoba (SECyT-UNC) and Consejo Nacional de

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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