cAMP response element-binding protein in the amygdala is required for long- but not short-term conditioned taste aversion memory

Abstract

In conditioned taste aversion (CTA) organisms learn to avoid a taste if the first encounter with that taste is followed by transient poisoning. The neural mechanisms that subserve this robust and long-lasting association of taste and malaise have not yet been elucidated, but several brain areas have been implicated in the process, including the amygdala. In this study we investigated the role of amygdala in general, and the cAMP response element-binding protein (CREB) in the amygdala in particular, in CTA learning and memory. Toward that end, we combined antisense technology in vivo with behavioral, molecular, and histochemical analysis. Local microinjection of phosphorothioate-modified oligodeoxynucleotides (ODNs) antisense to CREB into the rat amygdala several hours before CTA training transiently reduced the level of CREB protein during training and impaired CTA memory when tested 3-5 d later. In comparison, sense ODNs had no effect on memory. The effect of antisense was not attributable to differential tissue damage and was site-specific. CREB antisense in the amygdala had no effect on retrieval of CTA memory once it had been formed, and did not affect short-term CTA memory. We propose that the amygdala, specifically the central nucleus, is required for the establishment of long-term CTA memory in the behaving rat; that the process involves long-term changes, subserved by CRE-regulated gene expression, in amygdala neurons; and that the amygdala may retain some CTA-relevant information over time rather than merely modulating the gustatory trace during acquisition of CTA.

Fig. 1.

Sphere of diffusion of microinjected CREB antisense ODNs into the amygdala (A) and reduction in CREB-positive nuclei by CREB antisense (B–D). A, FITC-labeled CREB antisense was microinjected, and the fluorescence image recorded as detailed in Materials and Methods. CeM, Central amygdaloid nucleus, medial division; CeL, central amygdaloid nucleus, lateral division; BMA, basomedial amygdaloid nucleus, anterior part; BLA, basolateral amygdaloid nucleus, anterior part; Pir, piriform cortex. The microinjection cannula track and the lesion induced by the tip of the cannula are seen within the sphere of FITC diffusion. The schematic anatomical map is adapted from that of Paxinos and Watson (1986).B, The number of CREB-positive nuclei in the amygdala of CREB antisense (AS) versus sense (S) microinjected rats. In both groups CREB antisense was microinjected into the amygdala 14 hr before CTA training; n = 4 in each group. Theasterisk indicates significance for pair comparison in which a Scheffe contrast test was used with an α of 0.05. C, D, Immunohistochemistry of CREB using CREB antibodies in CREB sense (C) and antisense (D) microinjected rats. The selected fields were taken from the region of the CeL, indicated in A; dot, edge of the microinjection cannula-induced lesion.

Fig. 2.

Impairment of CTA by ODNs antisense to CREB in the amygdala. Aversion indices are plotted versus the test day. In each test day, the left (shaded)bar depicts the aversion index of normal CTA control animals (n = 23); the center(solid) bar depicts the aversion index of animals locally microinjected into the amygdala with antisense ODNs (n = 16); and the right(open) bar shows the index of animals receiving sense ODNs (n = 11). All microinjections into the amygdala were performed 14 hr before CTA training, as detailed in Materials and Methods. Inset, Spatial and temporal specificity of the antisense effect. The shaded bardepicts the result for normal controls; the hatched barsshow the results for rats microinjected with antisense.A, Normal CTA animals; B, antisense microinjected 14 hr before training 2 mm above the stereotaxic coordinates used for injection into the amygdala; C, antisense microinjected into the amygdala 14 hr before the first memory test, i.e., 36 hr after the completion of training; D, antisense microinjected into the amygdala 7 d before CTA training;n = 6 animals in each group.Asterisks indicate significance for pair comparison in which a Scheffe contrast test was used with an α of 0.05.

Fig. 3.

CREB antisense in the amygdala selectively impairs long- but not short-term memory. Aversion indices are plotted versus the time of test after CTA training. Closed circles, Rats microinjected with CREB antisense ODNs 14 hr before CTA training;open circles, control animals injected with saline instead of CREB antisense. The figure also depicts the time windows of LOB behavior (dark gray) and rearing (light gray), i.e., measures of the on-line effect of the malaise inducing-agent as a function of the time after LiCl injection intraperitoneally. For further details see Results. Theasterisk indicates significance for pair comparison in which a Scheffe contrast test was used with an α of 0.05.