eIF2α controls memory consolidation via excitatory and somatostatin neurons.

An important tenet of learning and memory is the notion of a molecular switch that promotes the formation of long-term memory1-4. The regulation of proteostasis is a critical and rate-limiting step in the consolidation of new memories5-10. One of the most effective and prevalent ways to enhance memory is by regulating the synthesis of proteins controlled by the translation initiation factor eIF211. Phosphorylation of the α-subunit of eIF2 (p-eIF2α), the central component of the integrated stress response (ISR), impairs long-term memory formation in rodents and birds11-13. By contrast, inhibiting the ISR by mutating the eIF2α phosphorylation site, genetically11 and pharmacologically inhibiting the ISR kinases14-17, or mimicking reduced p-eIF2α with the ISR inhibitor ISRIB11, enhances long-term memory in health and disease18. Here we used molecular genetics to dissect the neuronal circuits by which the ISR gates cognitive processing. We found that learning reduces eIF2α phosphorylation in hippocampal excitatory neurons and a subset of hippocampal inhibitory neurons (those that express somatostatin, but not parvalbumin). Moreover, ablation of p-eIF2α in either excitatory or somatostatin-expressing (but not parvalbumin-expressing) inhibitory neurons increased general mRNA translation, bolstered synaptic plasticity and enhanced long-term memory. Thus, eIF2α-dependent mRNA translation controls memory consolidation via autonomous mechanisms in excitatory and somatostatin-expressing inhibitory neurons.

Activity of Insula to Basolateral Amygdala Projecting Neurons is Necessary and Sufficient for Taste Valence Representation.

Conditioned taste aversion (CTA) is an associative learning paradigm, wherein consumption of an appetitive tastant (e.g., saccharin) is paired to the administration of a malaise-inducing agent, such as intraperitoneal injection of LiCl. Aversive taste learning and retrieval require neuronal activity within the anterior insula (aIC) and the basolateral amygdala (BLA). Here, we labeled neurons of the aIC projecting to the BLA in adult male mice using a retro-AAV construct and assessed their necessity in aversive and appetitive taste learning. By restricting the expression of chemogenetic receptors in aIC-to-BLA neurons, we demonstrate that activity within the aIC-to-BLA projection is necessary for both aversive taste memory acquisition and retrieval, but not for its maintenance, nor its extinction. Moreover, inhibition of the projection did not affect incidental taste learning per se, but effectively suppressed aversive taste memory retrieval when applied either during or before the encoding of the unconditioned stimulus for CTA (i.e., malaise). Remarkably, activation of the projection after novel taste consumption, without experiencing any internal discomfort, was sufficient to form an artificial aversive taste memory, resulting in strong aversive behavior upon retrieval. Our results indicate that aIC-to-BLA projecting neurons are an essential component in the ability of the brain to associate taste sensory stimuli with body states of negative valence and guide the expression of valence-specific behavior upon taste memory retrieval.SIGNIFICANCE STATEMENT In the present study we subjected mice to the conditioned taste aversion paradigm, where animals learn to associate novel taste with malaise (i.e., assign it negative valence). We show that activation of neurons in the anterior insular cortex (aIC) that project into the basolateral amygdala (BLA) in response to conditioned taste aversion is necessary to form a memory for a taste of negative valence. Moreover, artificial activation of this pathway (without any feeling of pain) after the sampling of a taste can also lead to such associative memory. Thus, activation of aIC-to-BLA projecting neurons is necessary and sufficient to form and retrieve aversive taste memory.

Intrinsic Excitability in Layer IV-VI Anterior Insula to Basolateral Amygdala Projection Neurons Correlates with the Confidence of Taste Valence Encoding.

Avoiding potentially harmful, and consuming safe food is crucial for the survival of living organisms. However, the perceived valence of sensory information can change following conflicting experiences. Pleasurability and aversiveness are two crucial parameters defining the perceived valence of a taste and can be impacted by novelty. Importantly, the ability of a given taste to serve as the conditioned stimulus (CS) in conditioned taste aversion (CTA) is dependent on its valence. Activity in anterior insula (aIC) Layer IV-VI pyramidal neurons projecting to the basolateral amygdala (BLA) is correlated with and necessary for CTA learning and retrieval, as well as the expression of neophobia toward novel tastants, but not learning taste familiarity. Yet, the cellular mechanisms underlying the updating of taste valence representation in this specific pathway are poorly understood. Here, using retrograde viral tracing and whole-cell patch-clamp electrophysiology in trained mice, we demonstrate that the intrinsic properties of deep-lying Layer IV-VI, but not superficial Layer I-III aIC-BLA neurons, are differentially modulated by both novelty and valence, reflecting the subjective predictability of taste valence arising from prior experience. These correlative changes in the profile of intrinsic properties of LIV-VI aIC-BLA neurons were detectable following both simple taste experiences, as well as following memory retrieval, extinction learning, and reinstatement.

Insula to mPFC reciprocal connectivity differentially underlies novel taste neophobic response and learning in mice.

To survive in an ever-changing environment, animals must detect and learn salient information. The anterior insular cortex (aIC) and medial prefrontal cortex (mPFC) are heavily implicated in salience and novelty processing, and specifically, the processing of taste sensory information. Here, we examined the role of aIC-mPFC reciprocal connectivity in novel taste neophobia and memory formation, in mice. Using pERK and neuronal intrinsic properties as markers for neuronal activation, and retrograde AAV (rAAV) constructs for connectivity, we demonstrate a correlation between aIC-mPFC activity and novel taste experience. Furthermore, by expressing inhibitory chemogenetic receptors in these projections, we show that aIC-to-mPFC activity is necessary for both taste neophobia and its attenuation. However, activity within mPFC-to-aIC projections is essential only for the neophobic reaction but not for the learning process. These results provide an insight into the cortical circuitry needed to detect, react to- and learn salient stimuli, a process critically involved in psychiatric disorders.

Parvalbumin interneuron inhibition onto anterior insula neurons projecting to the basolateral amygdala drives aversive taste memory retrieval.

Memory retrieval refers to the fundamental ability of organisms to make use of acquired, sometimes inconsistent, information about the world. Although memory acquisition has been studied extensively, the neurobiological mechanisms underlying memory retrieval remain largely unknown. Conditioned taste aversion (CTA) is a robust associative paradigm, through which animals can be trained to express aversion toward innately appetitive tastants. The anterior insula (aIC) is indispensable in the ability of mammals to retrieve associative information regarding tastants that have been previously linked with gastric malaise. Here, we show that CTA memory retrieval promotes cell-type-specific activation in the aIC. Using chemogenetic tools in the aIC, we found that CTA memory acquisition requires activation of excitatory neurons and inhibition of inhibitory neurons, whereas retrieval necessitates activation of both excitatory and inhibitory aIC circuits. CTA memory retrieval at the aIC activates parvalbumin (PV) interneurons and increases synaptic inhibition onto activated pyramidal neurons projecting to the basolateral amygdala (aIC-BLA). Unlike innately appetitive taste memory retrieval, CTA retrieval increases synaptic inhibition onto aIC-BLA-projecting neurons that is dependent on activity in aIC PV interneurons. PV aIC interneurons coordinate CTA memory retrieval and are necessary for its dominance when conflicting internal representations are encountered over time. The reinstatement of CTA memories following extinction is also dependent on activation of aIC PV interneurons, which increase the frequency of inhibition onto aIC-BLA-projecting neurons. This newly described interaction of PV and a subset of excitatory neurons can explain the coherency of aversive memory retrieval, an evolutionary pre-requisite for animal survival.

The Insula and Taste Learning.

The sense of taste is a key component of the sensory machinery, enabling the evaluation of both the safety as well as forming associations regarding the nutritional value of ingestible substances. Indicative of the salience of the modality, taste conditioning can be achieved in rodents upon a single pairing of a tastant with a chemical stimulus inducing malaise. This robust associative learning paradigm has been heavily linked with activity within the insular cortex (IC), among other regions, such as the amygdala and medial prefrontal cortex. A number of studies have demonstrated taste memory formation to be dependent on protein synthesis at the IC and to correlate with the induction of signaling cascades involved in synaptic plasticity. Taste learning has been shown to require the differential involvement of dopaminergic GABAergic, glutamatergic, muscarinic neurotransmission across an extended taste learning circuit. The subsequent activation of downstream protein kinases (ERK, CaMKII), transcription factors (CREB, Elk-1) and immediate early genes (c-fos, Arc), has been implicated in the regulation of the different phases of taste learning. This review discusses the relevant neurotransmission, molecular signaling pathways and genetic markers involved in novel and aversive taste learning, with a particular focus on the IC. Imaging and other studies in humans have implicated the IC in the pathophysiology of a number of cognitive disorders. We conclude that the IC participates in circuit-wide computations that modulate the interception and encoding of sensory information, as well as the formation of subjective internal representations that control the expression of motivated behaviors.