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The study of experimental extinction and of the spontaneous recovery of the extinguished memory could cast light on neurobiological mechanisms by which internal representations compete to control behavior. In this work, we use a combination of behavioral and molecular methods to dissect subprocesses of experimental extinction of conditioned taste aversion (CTA). Extinction of CTA becomes apparent only 90 min after the extinction trial. This latency is insensitive to muscarinic and beta-adrenergic modulation and to protein synthesis inhibition in the insular cortex (IC). Immediately afterwards, however, the extinguishing trace becomes sensitive to beta-adrenergic blockade and protein synthesis inhibition. The subsequent kinetics and magnitude of extinction depend on whether a spaced or massed extinction protocol is used. A massed protocol is highly effective in the short run, but results in apparent stagnation of extinction in the long-run, which conceals fast spontaneous recovery of the preextinguished trace. This recovery can be truncated by a beta-adrenergic agonist or a cAMP analog in the insular cortex, suggesting that spontaneous overtaking of the behavioral control by the original association is regulated at least in part by beta-adrenergic input, probably operating via the cAMP cascade, long after the offset of the conditioned stimulus. Hence, the performance of the subject in experimental extinction is the sum total of multiple, sometimes conflicting, time-dependent processes.

Taste consumption activates the extracellular responsive kinases 1-2 (ERK1-2) and the transcription factor Elk-1 in the insular cortex (IC) of the behaving rat. ERKs activation, an obligatory step for the encoding of long- but not short-term memory, was shown to be regulated by multiple neurotransmitter systems in the IC. Here I show, by the use of local microinfusions of pharmacological agents into the IC of the behaving rat, that a set of similar systems is required for the taste-induced activation of Elk-1. N-Methyl-D-aspartate (NMDA), glutamate metabotropic (mGlu), ionotropic AMPA/kainate (AMPA), muscarinic, and dopaminergic receptors, which all contribute to the acquisition of taste memory, are also responsible for Elk-1 activation. However, blockade of the beta-adrenergic transmission does not affect Elk-1 activation. I also show that the basal level of Elk-1 activation in cortex is mainly maintained by GABAergic transmission. Thus, the formation of taste memory triggers the activation of Elk-1 in the IC of the behaving rat via selected neurotransmitter and neuromodulatory systems.

In the behaving rat, the consumption of an unfamiliar taste activates the extracellular signal-regulated kinase 1-2 (ERK1-2) in the insular cortex, which contains the taste cortex. In contrast, consumption of a familiar taste has no effect. Furthermore, activation of ERK1-2, culminating in modulation of gene expression, is obligatory for the encoding of long-term, but not short-term, memory of the new taste (Berman et al., 1998). Which neurotransmitter and neuromodulatory systems are involved in the activation of ERK1-2 by the unfamiliar taste and in the long-term encoding of the new taste information? Here we show, by the use of local microinjections of pharmacological agents to the insular cortex in the behaving rat, that multiple neurotransmitters and neuromodulators are required for encoding of taste memory in cortex. However, these systems vary in the specificity of their role in memory acquisition and in their contribution to the activation of ERK1-2. NMDA receptors, metabotropic glutamate receptors, muscarinic, and beta-adrenergic and dopaminergic receptors, all contribute to the acquisition of the new taste memory but not to its retrieval. Among these, only NMDA and muscarinic receptors specifically mediate taste-dependent activation of ERK1-2, whereas the beta-adrenergic function is independent of ERK1-2, and dopaminergic receptors regulate also the basal level of ERK1-2 activation. The data are discussed in the context of postulated novelty detection circuits in the central taste system.

Rats were given to drink an unfamiliar taste solution under conditions that result in long-term memory of that taste. The insular cortex, which contains the taste cortex, was then removed and assayed for activation of mitogen-activated protein kinase (MAPK) cascades by using antibodies to the activated forms of various MAPKs. Extracellular responsive kinase 1-2 (ERK1-2) in the cortical homogenate was significantly activated within <30 min of drinking the taste solution, without alteration in the total level of the ERK1-2 proteins. The activity subsided to basal levels within <60 min. In contrast, ERK1-2 was not activated when the taste was made familiar. The effect of the unfamiliar taste was specific to the insular cortex. Jun N-terminal kinase 1-2 (JNK1-2) was activated by drinking the taste but with a delayed time course, whereas the activity of Akt kinase and p38MAPK remained unchanged. Elk-1, a member of the ternary complex factor and an ERK/JNK downstream substrate, was activated with a time course similar to that of ERK1-2. Microinjection of a reversible inhibitor of MAPK/ERK kinase into the insular cortex shortly before exposure to the novel taste in a conditioned taste aversion training paradigm attenuated long-term taste aversion memory without significantly affecting short-term memory or the sensory, motor, and motivational faculties required to express long-term taste aversion memory. It was concluded that ERK and JNK are specifically and differentially activated in the insular cortex after exposure to a novel taste, and that this activation is required for consolidation of long-term taste memory.

We demonstrate that the NMDA receptor is involved in taste learning in the insular cortex of the behaving rat and describe two facets of this involvement. Blockage of the NMDA receptor in the insular cortex by the reversible antagonist APV during training in a conditioned taste aversion (CTA) paradigm impaired CTA memory, whereas blockage of the NMDA receptor in an adjacent cortex or before a retrieval test had no effect. When rats sampled an unfamiliar taste and hence learned about it, either incidentally or in the context of CTA training, the tyrosine phosphorylation of the NMDA receptor subunit 2B (NR2B) in the insular cortex was specifically increased. The level of tyrosine phosphorylation on NR2B was a function of the novelty of the taste stimulus and the quantity of the taste substance consumed, properties that also determined the efficacy of the taste stimulus as a conditioned stimulus in CTA; however, blockage of the NMDA receptor by APV during training did not prevent tyrosine phosphorylation of NR2B. We suggest that tyrosine phosphorylation of NR2B subserves encoding of saliency in the insular cortex during the first hours after an unfamiliar taste is sampled and that this encoding is independent of another, necessary role of NMDA receptors in triggering experience-dependent modifications in the insular cortex during taste learning. Because a substantial fraction of the NR2B protein in the insular cortex seems to be expressed in interneurons, saliency and the tyrosine phosphorylation of NR2B correlated with it may modulate inhibition in cortex.

We have recently shown that in the gustatory cortex of the rat, taste learning enhances protein tyrosine phosphorylation and taste memory is blocked by muscarinic antagonists. A major protein whose tyrosine phosphorylation is stimulated by taste learning in cortex is a 180 kDa synaptic glycoprotein identified as the NMDA receptor subunit 2B (NR2B). Here we report that microinjection of carbachol into the taste cortex modulates protein tyrosine phosphorylation similarly to the effect of unfamiliar taste, and that a 180 kDa protein whose tyrosine phosphorylation is enhanced in vivo by carbachol is NR2B. These data, combined with our previous findings, are in line with the hypothesis that muscarinic input plays a role in encoding new items in memory, and that tyrosine phosphorylation of NR2B is involved in this process.