ABSTRACT
Se presenta una revisión de distintos estudios que emplearon la técnica de AgNOR en el campo de las neurociencias del comportamiento. AgNOR es una técnica histológica que permite estudiar la activación cerebral de una región específica. Esta técnica ha sido utilizada en distintos grupos zoológicos que comprenden desde peces y anfibios hasta mamíferos, incluyendo actividad neural tanto en procesos normales como patológicos. En conjunto, la técnica de AgNOR tiene tres notables ventajas con respecto a otras técnicas histológicas como las inmunohistoquímicas. En primer lugar, su bajo costo. En segundo lugar, su robustez. Finalmente, la posibilidad de ser aplicada con facilidad a cualquier especie animal, siendo esto último particularmente relevante en el campo de la Psicología Comparada. Paradójicamente a pesar de estas ventajas, es una técnica poco conocida y empleada. El propósito de este artículo es contribuir a su difusión dentro de la comunidad de científicos del comportamiento.
In this paper we review the use of AgNOR technique in the field of behavioral neuroscience. AgNOR is a histological technique used to study the activation of a specific brain region. NORs (nucleolar organizer regions) are the cell nucleus compartments where part of the ribosomal synthesis occurs. When a neuron enhances its protein synthesis demand, for example during a learning process, NORs increase their size. Staining with silver nitrate, colors the cell nucleus in brown and the cell nucleolus in dark brown, nearly black. Thus, the size of the nucleus and the nucleolus can be easily measured in histological preparations treated with silver nitrate through any appropriated image processing software. In this manner, AgNOR technique consist in staining the tissue of subjects that have received different treatments and comparing the average size of the nucleolus of a particular brain region, which is an indirect marker of cellular activity. The use of AgNOR technique in the study of neural basis of learning, typically involves: (1) training animals in a particular task, (2) after acquisition, animals in the trained group along with animals from a control condition are sacrificed in order to obtain a brain sample, (3) brains of animals from both conditions are processed and compared. This procedure was successfully used to study telencephalic activation during spatial learning in fish (Carassius auratus). The task consisted in using visual cues to identify the correct exit from a trap. In the case of fish, the histological analysis showed an increase of the nucleolus size in the lateral pallium, proposed as homologous to the mammalian hippocampus, observing that this area is involved in spatial memory. In our laboratory this technique was also used to analyze the neural activity after spatial learning in the terrestrial toad Rhinella arenarum. We conducted several studies where dehydrated animals had to use visual cues to locate the correct position of a water source. In accordance with fishs data, our results indicated a higher activity of the hippocampal homologous region (the amphibian medial pallium), showing a high degree of functional equivalence. On the other hand, we also studied in toads the telencephalic activity during an aversive learning task. Here, it was observed an increase in the average of NORs size in the ventrolateral telencephalic region, the striatum (proposed as homologous to the mammalian amygdala), also showing a highly preserved functional equivalence. In mammals, AgNOR technique was used with different purposes, such as the study of changes in hippocampal activity during development in rats, the comparison of the basal activity of two regions of the rats hippocampus, the CA1 and the CA3, both strongly related with learning, or the the hippocampal activity in two different situations in which the liver activity was altered (by an artificial cholestasis, or by alcohol consumption). Finally, AgNOR technique was also used in the study of human brain activity of subjects that had committed suicide. On the whole, the AgNOR technique has three distinct advantages over other histological techniques such as the immunohistochemical. First, their low cost. Second, their robustness. Finally, the fact that it can be easily applied to any animal species, which makes it particularly attractive to the field of the Comparative Psychology. But paradoxically, despite these advantages it is a poorly known and used technique. The aim of this paper is to contribute to the diffusion of the AgNOR technique in the community of behavioral scientists, showing that its use has provided a valuable contribution to research in different fields and animal species.
ABSTRACT
En este artículo se describen desde una perspectiva comparativa los fenómenos de bloqueo, ensombrecimiento e inhibición latente, enfatizando su presencia en tareas de aprendizaje espacial. Estos fenómenos de aprendizaje, ampliamente observados en otras clases de vertebrados e invertebrados, han sido recientemente descriptos por primera vez en anfibios, un grupo de vertebrados con un cerebro filogenéticamente antiguo. Tomando como modelo al sapo terrestre Rhinella arenarum, se revisarán los tres fenómenos de aprendizaje asociativo mencionados en una situación de aprendizaje espacial: (1) bloqueo entre claves visuales asociadas a una meta, (2) ensombrecimiento de una clave visual lejana por la presencia de una clave cercana y (3) inhibición latente debida a la pre-exposición a una clave visual. Todos los entrenamientos se llevaron a cabo en una arena circular de color blanco, utilizando agua como recompensa. Dentro de la arena, se distribuyeron cuatro piletas de acrílico en forma de cruz contra las paredes laterales (sólo una tenía acceso a la recompensa). En las paredes interiores de la arena circular se colocaron varias señales visuales para guiar a los animales. Los resultados obtenidos en sapos indican que estos fenómenos, observados previamente en aves y mamíferos, también se encuentran en este grupo (utilizando un paradigma de aprendizaje espacial con claves visuales cercanas y lejanas). Este primer registro en anfibios sugiere que los mecanismos biológicos de estos fenómenos de aprendizaje han surgido muy tempranamente en el curso de la evolución de los vertebrados totalmente terrestres y que los mismos han sido fuertemente conservados. El análisis comparado de estos hallazgos contribuirá a mejorar el entendimiento de los mecanismos biológicos que subyacen al aprendizaje espacial, en busca de patrones funcionales comunes con otras clases de vertebrados y potencialmente presentes en un ancestro común.
This article describes blocking, overshadowing and latent inhibition phenomena from a comparative perspective, emphasizing their presence in spatial learning tasks. These learning phenomena, previously observed in other vertebrates and invertebrates classes, have been recently reported for the first time in amphibians, a vertebrate group with a phylogenetically ancient brain. We use the terrestrial toad Rhinella arenarum as animal model to analyze the three mentioned associative phenomena in a spatial learning situation: (1) blocking between visual cues associated to a goal, (2) overshadowing of a distant visual cue by the presence of a nearby cue, and (3) latent inhibition generated by the pre-exposure to a visual cue. All trainings were conducted in a white circular arena, using water as reward. Inside the arena, four green acrylic container were distributed in a cross shape against the side walls (only one with access to reward). On the inner walls of the circular arena they were placed several visual cues for guiding animals. In all studies described in this article toads were partially dehydrated to motivate them to search for water. In the blocking situation, experimental animals had the rewarded container signaled by a visual cue on the wall above the container. In the second phase of training other visual cue was added. The results revealed that in these animals the prior training with only one of the visual cues blocked the association of the reward with the other cue, when both cues were then presented simultaneously to indicate the position of reward. In the overshadowing situation, experimental animals had from the beginning the rewarded container signaled by two visual cues on the wall, one to 10 cm right (named near cue), and the other placed approximately 70 cm to the left (between the adjacent pool and the opposite, named far cue). The results indicated that the location of a visual cue located away from reward was overshadowed by the presence of a nearby cue. Finally, in the situation of latent inhibition, animals of pre-exposed group had five previous training sessions, where a visual cue was presented without reward. Then, when in the subsequent training this visual cue signaled the reward, animals pre-exposed needed more sessions to reach the asymptote of learning compared to other non-pre-exposed animals. Therefore, the pre-exposure to the visual cue (i.e., unreinforced exposure to the stimulus) significantly retarded the acquisition, delaying the association of this cue with the reward. Taking into account that these phenomena have been observed previously in birds and mammals, this first record in amphibians (using a spatial learning paradigm with near and far visual cues) suggests that biological mechanisms of these learning phenomena have emerged very early in the course of the evolution of fully terrestrial vertebrates and that they have been strongly preserved. With regard to the underlying neural substrates, the relationship of the hippocampal formation with the processes of learning and spatial memory is a constant in all vertebrate species studied. Analyzing the particular case of amphibians, compared with other groups of tetrapods, their telencephalon has a simple organization (in the evolutionary sense non-derived). In this aspect, the medial pallium, region in the dorso-medial quadrant of the hemisphere, is considered homologous to the mammalian hippocampal formation (based on their topographic position, its interconnections with other telencephalic areas and neurohistochemical data). Heretofore, the dependence of spatial learning with the functioning of the medial cortex was thought to be a primitive character of amniotes. However, this feature can now be extended to the group of amphibians, as recently has been observed that the lesion of the medial pallium impairs spatial learning. Thus, the evidence collected until this moment in amphibians suggests that this relationship may have emerged earlier than previously thought. On the whole, the comparative analysis of these findings will contribute to a better understanding of the biological mechanisms underlying spatial learning, thereby looking for common functional patterns with other vertebrate classes, potentially present in a common ancestor.