Long-term memory is facilitated by cAMP response element-binding protein overexpression in the amygdala

Abstract

At least two temporally and mechanistically distinct forms of memory are conserved across many species: short-term memory that persists minutes to hours after training and long-term memory (LTM) that persists days or longer. In general, repeated training trials presented with intervening rest intervals (spaced training) is more effective than massed training (the same number of training trials presented with no or short intervening rest intervals) in producing LTM. LTM requires de novo protein synthesis, and cAMP response element-binding protein (CREB) may be one of the transcription factors regulating the synthesis of new proteins necessary for the formation of LTM. Here we show that rats given massed fear conditioning training show no or weak LTM, as measured by fear-potentiated startle, compared with rats given the same amount of training but presented in a spaced manner. Increasing CREB levels specifically in the basolateral amygdala via viral vector-mediated gene transfer significantly increases LTM after massed fear training. The enhancing effect of CREB overexpression on LTM formation is shown to be specific in terms of biochemistry, anatomy, time course, and the training procedure used. These results suggest that CREB activity in the amygdala serves as a molecular switch for the formation of LTM in fear conditioning.

Fig. 1.

Effect of ITI on LTM as assessed by fear-potentiated startle. Fear-potentiated startle (FPS) difference scores were calculated by subtracting the mean startle amplitudes obtained on noise-alone trials from the mean startle amplitudes obtained on light–noise trials and used as an index of LTM. The mean ± SEM level of LTM is shown after training, which consisted of four light–shock pairings with ITIs of 3 sec, 5 sec, 10 sec, 15 sec, 2 min, and 8 min. Massed training (10 sec and less) produced very weak LTM (∼50 units), whereas spaced training (8 min) produced robust LTM.

Fig. 2.

Immunocytochemical examination of the basolateral complex of the amygdala and caudate nucleus after infusion of HSV vectors. A, Expression of LacZ 3 d after infusion of HSV-LacZ into the basolateral amygdala. Sections are counterstained with neutral red and presented at low magnification. B, Expression of CREB 3 d after control infusion of HSV-LacZ into the basolateral amygdala. C, Overexpression of CREB 3 d after infusion of HSV-CREB into the caudate nucleus. D, Overexpression of CREB 3 d after infusion of HSV-CREB into the basolateral amygdala. * indicates location of infusion.ic, Internal capsule; opt, optic tract;Ce, central nucleus of the amygdala; L, lateral nucleus of the amygdala; BL, basolateral nucleus of the amygdala; BM, basomedial nucleus of the amygdala;Pir, pyriform cortex; ec, external capsule; CP, caudate putamen. Scale bars, 0.2 mm.

Fig. 3.

Effect of infusion of HSV vectors into the basolateral amygdala and extra-amygdala areas on the subsequent LTM (mean ± SEM) observed 48 hr after massed fear conditioning training (10 sec ITI). HSV vectors and PBS infusions were aimed at the basolateral amygdala and a control region (the caudate nucleus). However, several infusions of HSV-CREB missed the amygdala target region and are thus designated as extra-amygdala (HSV-CREB Extra-Amygdala). Rats infused with HSV-CREB into the basolateral amygdala showed significantly greater LTM than rats similarly infused with PBS, HSV-LacZ, or HSV-mCREB or rats infused with HSV-CREB into brain regions surrounding the amygdala or directly into the caudate nucleus.

Fig. 4.

Specificity of the LTM-enhancing effects of CREB overexpression in the amygdala on LTM formation after massed training (10 sec ITI). A, Similar reactions to footshock were observed during training in rats infused with PBS, HSV-LacZ, HSV-CREB, or HSV-mCREB into the basolateral amygdala. B, Effects of HSV vectors on explicitly unpaired massed training. Explicitly unpaired conditioning fails to produce LTM in unoperated control rats. Furthermore, intrabasolateral amygdala infusion of PBS or HSV-CREB does not enhance LTM after unpaired massed training. C, Time course of the effect of HSV vectors on LTM formation after massed training. Animals that received HSV-CREB 3 d before massed training show greater LTM when retested 14 d after infusion than animals similarly treated with HSV-LacZ or animals given HSV-CREB 14 d before massed training and tested 48 hr later.

Fig. 5.

Time course of transgene expression after infusion of HSV-LacZ. A, High expression of the LacZ 3 d after infusion of HSV-LacZ into the basolateral amygdala. Sections are counterstained with neutral red and presented at low magnification.B, Low expression of the LacZ transgene 14 d after infusion of HSV-LacZ into the basolateral amygdala.

Fig. 6.

LTM after spaced (8 min ITI) training using different intensities of footshock after infusion of HSV vectors into the basolateral amygdala. A, High-footshock intensity (0.6 mA). The high levels of LTM 48 hr after spaced training were not significantly different in control (unoperated) rats and those receiving PBS or HSV-CREB into the basolateral amygdala.B, Low-footshock intensity (0.3 mA). The modest levels of LTM were not significantly different in control (unoperated) rats and those receiving infusions of PBS or HSV-CREB into the basolateral amygdala. The higher footshock intensity (0.6 mA) produced significantly greater LTM that did the weaker footshock intensity (0.3 mA), but increasing CREB levels did not change this.

Fig. 7.

STM, as measured by fear-potentiated startle (FPS) difference scores, after massed training in animals infused with HSV vectors in the basolateral amygdala. Control rats that received unpaired massed presentation of the light and shock show less STM than rats that had received infusion of PBS and paired massed training. However, rats infused with HSV-CREB did not differ in STM levels from rats infused with PBS.