Acquisition, consolidation, reconsolidation, and extinction of eyelid conditioning responses require de novo protein synthesis - PubMed (original) (raw)

Acquisition, consolidation, reconsolidation, and extinction of eyelid conditioning responses require de novo protein synthesis

Mari Carmen Inda et al. J Neurosci. 2005.

Abstract

Memory, as measured by changes in an animal's behavior some time after learning, is a reflection of many processes. Here, using a trace paradigm, in mice we show that de novo protein synthesis is required for acquisition, consolidation, reconsolidation, and extinction of classically conditioned eyelid responses. Two critical periods of protein synthesis have been found: the first, during training, the blocking of which impaired acquisition; and the second, lasting the first 4 h after training, the blocking of which impaired consolidation. The process of reconsolidation was sensitive to protein synthesis inhibition if anisomycin was injected before or just after the reactivation session. Furthermore, extinction was also dependent on protein synthesis, following the same temporal course as that followed during acquisition and consolidation. This last fact reinforces the idea that extinction is an active learning process rather than a passive event of forgetting. Together, these findings demonstrate that all of the different stages of memory formation involved in the classical conditioning of eyelid responses are dependent on protein synthesis.

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Figures

Figure 1.

Anisomycin transiently inhibits protein synthesis. A, Representation of KA and anisomycin administration. KA was always injected 30 min before the animal was killed, whereas anisomycin was administered at variable times (indicated by shaded arrows) with respect to KA injection (indicated by the filled arrow). Negative values on the timeline indicate anisomycin administration before KA; 0 indicates simultaneous administration; and positive values represent administration after KA injection (n = 3 per group). B, Representative hippocampal c-Fos protein immunohistochemistry obtained from anisomycin alone-treated (Aniso), KA alone-treated (Sal + KA), and anisomycin- and KA-treated animals. The time between anisomycin and KA injections is indicated in the top right corner. C, Quantitative analysis of c-Fos expression in brain areas related to processes of learning and memory: hippocampus, piriform cortex, and amygdala. Also, the protein inhibition index is shown. CA1, CA1 field; CA3, CA3 field; DG, dentate gyrus; Hp, hippocampus; Pir Cx, piriform cortex; Amyg, amygdala nucleus; Sal, saline. Scale bar, 500 μm.

Figure 2.

The morphology of motor, hippocampal, and cerebellar cortices was not altered by anisomycin injection. a-f, NeuN immunostaining of the motor, hippocampal, and cerebellar cortices from saline-injected (a-c) and anisomycin-injected (d-f) animals (magnification, 5×). g-j, GFAP immunostaining of the hippocampus obtained from saline-injected (g, h) and anisomycin-injected (i, j) mice. As illustrated at 20× magnification, there was no glial reaction in the CA3 layer in saline-injected (h) or anisomycin-injected (j) animals. k-n, Calbindin immunostaining of the hippocampal and cerebellar cortices of saline-injected (k, l) and anisomycin-injected (m, n) mice. The lack of anisomycin-induced changes in the mossy fiber tract is evidenced by the absence of any noticeable difference in the staining of the stratum lucidum (CA3 field) between saline-treated (k) and anisomycin-treated (m) mice (magnification, 20×). Note the intactness of the Purkinje cell dendritic arborization in the molecular layer of the cerebellum after anisomycin treatment (n) compared with saline-treated animals (l). CA1, CA1 field; CA3, CA3 field; DG, dentate gyrus; PCL, Purkinje cell layer; MF, mossy fiber tract. In all cases, n = 3 per group.

Figure 3.

Anisomycin administration does not affect the electrical activity of the OOM. A, Representative EMG records from the OOM showing R1 and R2 components of a reflex blink evoked by the electrical stimulation of the supraorbitary branch of the trigeminal nerve in saline- and anisomycin-injected mice. B, C, Graphs representing the mean ± SEM of the latency (B) and voltage amplitude (C) of R1 and R2 components during habituation and during the fourth and eighth conditioning sessions in saline-injected (Sal; open bars) and anisomycin-injected (Aniso; filled bars) animals (n = 20 per group). H, Habituation; C4, C8, fourth and eighth conditioning sessions, respectively.

Figure 4.

A weak-strong electrical shock of the supraorbitary branch of the trigeminal nerve, with a trace paradigm, was used for classical conditioning of eyelid responses. A, Differential progress in the percentage of CRs per session across conditioning between conditioned (Cond.; filled circles) and pseudoconditioned (Pseudo.; open circles) groups (n = 8 per group). B, C, Representative single-trial records of the EMG of the OOM during different training sessions obtained from one subject of the conditioned (B) and pseudoconditioned (C) groups. H, Habituation; C1-C8, first through eighth conditioning sessions; PC8, eighth pseudoconditioning session.

Figure 5.

Two critical periods of protein synthesis are required for acquisition and consolidation of eyelid conditioned responses. A, Diagram illustrating the two phases of the experiment. Phase 1 consisted of eight conditioning sessions. During each session, 60 CS-US trials were presented. In this phase, either 100 mg/kg anisomycin (Aniso; experimental subjects) or saline (Sal; control subjects) was injected at variable times from the beginning of each session: -30 and +30 min and +4 h. Because all saline groups were indistinguishable, their results were grouped for the sake of simplicity. For phase 2, the above-mentioned groups received seven additional training sessions 10 d after the end of phase 1; however, neither anisomycin nor saline was administered during these additional sessions. B, C, Acquisition (phase 1) and retention (phase 2) of eyelid CRs, expressed as percentage of response (mean ± SEM). Filled circles, Saline-injected animals; open circles, triangles, squares, mice injected with anisomycin at -30 and +30 min and +4 h, respectively (n = 8 per group). D, The retention index represents the ratio between the percentage of CRs during the first training session of phase 2 and the last training session of phase 1. H, Habituation; C1-C8, first through eighth conditioning sessions; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 6.

Anisomycin administration before (-30 min) or after (+30 min) reactivation led to impaired reconsolidation. A, Diagram illustrating the experimental design. For phase 1, eight training sessions were performed. For phase 2 (i.e., 10 d after phase 1), a single training session [reactivation (RA)] was performed. Either anisomycin (experimental subjects) or saline (control subjects) was injected at -30 or +30 min for the reactivation session (n = 8 per group). Because results were similar in the two anisomycin-treated groups, only data corresponding to the +30 min group are shown. Phase 3 was performed to evaluate reconsolidation. For this, animals underwent training sessions 6 h (to test STM) and 48 h (to test LTM) after the end of the reactivation session. During each session, 60 CS-US trials were presented. B, Percentage of responses (mean ± SEM) during habituation, last conditioning, reactivation, STM, and LTM sessions. Open circles, Group injected with anisomycin at +30 min; filled circles, consolidated saline groups. C, Diagram illustrating an additional experimental design similar to that described in A. In this case, anisomycin or saline was injected at +4 h, and only LTM was tested (n = 8 per group). D, Percentage of responses (mean ± SEM) during habituation, last conditioning, reactivation, and LTM sessions. Open squares, Group injected with anisomycin at +4 h; filled squares, saline group. Cond, Conditioning; Aniso, anisomycin; H, habituation; C8, eighth conditioning session; Sal, saline; **p ≤ 0.01.

Figure 7.

Acquisition and consolidation of extinction require protein synthesis. A, Diagram illustrating the experimental design. Phase 1 was performed as illustrated in Figure 5_A_. For phase 2 (i.e., extinction), four CS-alone sessions were performed. During each extinction session, 60 CS-alone trials were presented. Either 100 mg/kg anisomycin (experimental subjects) or saline (control subjects) was injected at variable times from the beginning of each session: -30 and +30 min and +4 h (n = 8 per group). Because all saline groups were indistinguishable, their results were grouped for the sake of simplicity. For phase 3, the above-described groups received an additional CS-alone session [i.e., reactivation (RA)] 10 d after the end of phase 2. B, Percentage of responses (mean ± SEM) during habituation, eight conditioning, extinction, and reactivation sessions. Filled circles, Saline-injected animals; open circles, triangles, squares, mice injected with anisomycin at -30 and +30 min and +4 h, respectively. C, Retention index represents the ratio between the first training session in phase 3 and the last training session in phase 2. Cond, Conditioning; Aniso, anisomycin; H, habituation; C8, eighth conditioning session; Sal, saline; E1-E4, four extinction sessions; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 8.

Proposed model of experience-dependent gene expression in synaptic plasticity and memory consolidation processes. Synaptic activity driven by experience leads to alterations in intracellular second messenger levels, which in turn activate cellular kinases and phosphatases. These enzymes modulate the activity of a wide range of preexisting cellular proteins, including synaptic components and nuclear transcription factors. In the nucleus, the activation of transcription factors initiates a cascade of gene expression required for the formation of the long-term memory.

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Acquisition, consolidation, reconsolidation, and extinction of eyelid conditioning responses require de novo protein synthesis - PubMed (original) (raw)