Research report
Molecular signalling pathways in the cerebral cortex are required for retrieval of one-trial avoidance learning in rats

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Abstract

Rats were implanted bilaterally with cannulae in the CA1 region of the dorsal hippocampus, the entorhinal cortex, anterior cingulate cortex, posterior parietal cortex, or the basolateral complex of the amygdala. The animals were trained in one-trial step-down inhibitory avoidance and tested 24 h later. Prior (10 min) to the retention test, through the cannulae, they received 0.5 μl infusions of a vehicle (2% dimethylsulfoxide in saline), or of the following drugs dissolved in the vehicle: the glutamate NMDA receptor blocker, aminophosphonopentanoic acid (AP5, 2.0 or 5.0 μg), the AMPA receptor blocker, 6,7-dinitroquinoxaline-2,3 (1H,4H)dione (DNQX, 0.4 or 1.0 μg), the metabotropic receptor antagonist, methylcarboxyphenylglycine (MCPG, 0.5 or 2.5 μg), the inhibitor of cAMP-dependent protein kinase (PKA), Rp-cAMPs (0.1 or 0.5 μg), the PKA stimulant, Sp-cAMPs (0.5 μg), or the inhibitor of the mitogen-activated protein kinase (MAPK), PD098059 (10 or 50 μM). All these drugs, at the same doses, had been previously found to alter long-term memory formation of this task. Here, retrieval test performance was blocked by DNQX, MCPG, Rp-cAMPs and PD098059 and enhanced by Sp-cAMPs infused into CA1 or the entorhinal cortex. The drugs had similar effects when infused into the parietal or anterior cingulate cortex, except that in these two areas AP5 also blocked retrieval, and in the cingulate cortex DNQX had no effect. Infusions into the basolateral amygdala were ineffective except for DNQX, which hindered retrieval. None of the treatments that affected retrieval had any influence on performance in an open field or in a plus maze; therefore, their effect on retention testing can not be attributed to an influence on locomotion, exploration or anxiety. The results indicate that the four cortical regions studied participate actively in, and are necessary for, retrieval of the one-trial avoidance task. They require metabotropic and/or NMDA glutamate receptors and PKA and MAPK activity. In contrast, the basolateral amygdala appears to participate only through a maintenance of its regular excitatory transmission mediated by glutamate AMPA receptors.

Introduction

Retrieval of one-trial step-down avoidance is inhibited by the bilateral infusion into the dorsal CA1 area of the rat hippocampus by antagonists of AMPA/kainate or metabotropic glutamate receptors, or by inhibitors of the cAMP-dependent protein kinase (PKA), mitogen-activated protein kinase (MAPK) or protein kinase C (PKC) signalling pathways [20], [22], [44]. The effect of the drugs is not as a result of influences on locomotion, exploration or anxiety, because the treatments had no effect on behaviour in an open-field or a plus-maze [22], [44]. To our knowledge, these are the first data that provide hints on the molecular basis of retrieval. The involvement of the enzymatic pathways mentioned in CA1 is important because they are known to be crucial for consolidation of this task [2], [3], [17], [19], [20], [29], [31], [43], [44], [45] and others [2], [17], [29], as well as of long-term potentiation [17], [29]. Further, there is ample cross-talk between these signalling pathways [24], [29], [37], particularly PKA and MAPK [24], [29]. Glutamate receptors stimulate PKA and MAPK activity (see below) [17], [29]. AMPA, NMDA and metabotropic glutamate receptors in CA1 [6], [8], [15], [17], [19] are necessary for memory formation of this or other tasks.

The CA1 area of the hippocampus is part of a circuit that includes the subiculum, entorhinal cortex, dentate gyrus and CA3 [42]. This circuit has been recently demonstrated to be functionally active and to be capable of reverberation [12]. By virtue of the longitudinal spread of the connections of its various components [11], [20], it probably involves a large portion of the hippocampus every time it is activated. Recently it was calculated that 40% of the hippocampus is needed for encoding and 70% of it is needed for retrieval of a spatial learning task [30]. Measurement of the activity of PKA [3], PKC [31] or other enzymes [17] at various times after one-trial inhibitory avoidance training reveals marked changes measurable in the entire rat hippocampus.

The circuit described above is connected through the entorhinal region to many areas of the cortex, including the posterior parietal and cingulate cortex, and to the basolateral nuclear complex of the amygdala as well [11], [42], [47]. All these areas participate in the encoding and consolidation of one-trial inhibitory avoidance, each in a different manner [1], [5], [7], [17], [19], [21], [27]. The entorhinal, parietal and cingulate cortex are involved in consolidation through NMDA receptor-mediated, muscimol-sensitive mechanisms operating in a timed fashion in the first few hours of consolidation in parallel to hippocampal processing [21], [27]. The anterior cingulate seems to play a lesser role in consolidation of the one-trial avoidance task [27]; but there is some evidence that it might be relevant to at least some forms of retrieval in other tasks [39]. The basolateral amygdala is critical for the early modulation of consolidation of aversively-motivated experiences [7], [26], [42] and may be important for the expression of emotional components at the time of retrieval [14], [18], [23], [28], [47]. The entorhinal and parietal cortex are involved in the modulation of consolidation hours after training [1], [17]. Various studies point to the need of an intertwined activity of all these brain region, as well as others, in the consolidation of one-trial avoidance and related tasks [1], [5], [15], [17], [18], [20], [21], [42].

Here we study the effect on retrieval of one-trial avoidance of the bilateral infusion into CA1, entorhinal cortex, posterior parietal cortex, anterior cingulate cortex, and basolateral amygdala of drugs acting on specific molecular mechanisms known to be critical for encoding and consolidation of this task. The drugs used are: the glutamate AMPA receptor antagonist, 6,7-dinitroquinoxaline-2,3 (1H,4H)dione (DNQX); the glutamate NMDA receptor antagonist, DL-amino-5-phosphonopentanoic acid (AP5); the generic glutamate metabotropic receptor antagonist, α-methyl-(4-carboxyphenyl)glycine (MCPG); the inhibitor (Sp-cAMPs) and the stimulant (Sp-cAMPs) of the cAMP-dependent protein kinase (PKA); and the inhibitor of the mitogen-activated protein kinase (MAPK) pathway, PD098059. The choice of these particular drugs obeys to the following reasons. AMPA receptors mediate most of the regular excitatory synaptic transmission in the brain. NMDA and metabotropic receptors lead to an increase in cellular Ca2+; the former because of Ca2+ entry, and the latter through the release of endogenous Ca2+. The increased Ca2+ in turn stimulates a variety of metabolic processes, including the activity of the protein kinases mentioned [17], [29], [43], [44], [45]. Metabotropic receptors, in addition, can stimulate PKA indirectly via activation of adenylyl cyclase [41]. In CA1, PKA and MAPK are crucial to memory formation [1], [3], [17], [22], [44], [45], [46] and, as shown recently [20], [22], also for the retrieval of long-term memory.

Section snippets

Material and methods

Five experiments were carried out. In all of them, rats were trained in one-trial inhibitory avoidance and tested for retention 24 h later, a time at which long-term memory (LTM) is well established [17], [19]. Drugs were bilaterally infused through indwelling cannulae into different brain structures 10 min prior to retention testing. Experiment 1 studied the CA1 region. Experiment 2 investigated the entorhinal cortex. Experiment 3 examined the posterior parietal cortex. Experiment 4 studied

Experiment 1: effect on retrieval of drugs infused bilaterally into CA1

Training session latencies were not significantly different among groups in a super ANOVA (overall mean, 4.7 s; median, 4.5 s; range, 1.0–11.0 s) (N=119).

Retention test performance was significantly impaired by DNQX at the two dose levels studied, by MCPG at the higher dose, and by Rp-cAMPs and PD098059 at the two doses studied. In all these groups, training-test latency differences were not significant in Mann–Whitney U-tests at P=0.1 level. Retention test performance was significantly

Discussion

The results are clear and their interpretation is simple. The effect of the drugs studied is not explainable by alterations in locomotor or exploratory activity or anxiety levels. They may therefore be viewed as effects on retrieval itself. By the way, it may be noted that the variability of the effects of the various treatments on plus maze or open field behaviour appeared to be larger than that observed in the retention test of inhibitory avoidance. This further attests to the specificity of

Acknowledgements

Supported by PRONEX, Brazil. We thank C. Rodrigues, T. de David, C. Madche, A. Aguzzoli, D.M. Cardoso and B. Moletta for their technical help in the experiments.

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