Kazumi Takahashi

The IR-mediated neuronal activation (IRNA) technology allows stimulation of neurons in the brain that heterologously-express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the current protocol, a ligand against the complex consisting of IR84a and IR8a subunits, phenylacetic acid (PhAc), is locally injected into a brain region, because of a low efficiency of PhAc for the delivery into the brain across the blood-brain barrier. To circumvent this invasive injection, here we developed a strategy for activation of target neurons in the brain through peripheral administration with a precursor of PhAc, methyl ester of PhAc (PhAcM), which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. Peripheral administration of with PhAcM activated IR84a/IR8a-expressing neurons in the locus coeruleus of mice and increased the release of neurotransmitters in their nerve terminal regions. (S)-2-phenylpropionic acid ((S)-PhPr) was newly identified as a ligand for IR84a/IR8a, and peripheral administration with the methyl ester of PhPr with the S-configuration [(S)-PhPrM] caused similar effects on the target neurons. In addition, cell-type specific expression of IR84a/IR8a complex in the striatum of rats was unilaterally induced with a viral vector based on the Cre-loxP system. Peripheral administration with PhAcM or (S)-PhPrM stimulated the neurotransmitter release in the ipsilateral terminal regions of the vector-injected striatum, and PhAcM administration resulted in rotational behavior. Finally, we demonstrated that the metabolites of the peripherally administered-radiolabeled ( S )-PhPrM accumulated in the IR84a/IR8a-expressing region in the striatum of the vector-injected rats. These results demonstrate that the systemic IRNA technique provides a powerful strategy for remote manipulation of diverse types of target neurons in the mammalian central nervous system.

The ability of animals to retrieve memories stored in response to the environment is essential for behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. Here, we developed a novel chemogenetic activation strategy exploiting insect olfactory ionotropic receptors (IRs), termed “IR-mediated neuronal activation,” and used it for selective stimulation of NE neurons in the locus coeruleus (LC).Drosophila melanogasterIR84a and IR8a subunits were expressed in LC NE neurons in transgenic mice. Application of phenylacetic acid (a specific ligand for the IR84a/IR8a complex) at appropriate doses induced excitatory responses of NE neurons expressing the receptors in both slice preparations andin vivoelectrophysiological conditions, resulting in a marked increase of NE release in the LC nerve terminal regions (male and female). Ligand-induced activation of LC NE neurons enhanced the retrieval process of conditioned taste aversion without affecting taste sensitivity, general arousal state, and locomotor activity. This enhancing effect on taste memory retrieval was mediated, in part, through α₁- and β-adrenergic receptors in the basolateral nucleus of the amygdala (BLA; male). Pharmacological inhibition of LC NE neurons confirmed the facilitative role of these neurons in memory retrieval via adrenergic receptors in the BLA (male). Our findings indicate that the LC NE system, through projections to the BLA, controls the retrieval process of taste associative memory.SIGNIFICANCE STATEMENTNorepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation, but the role of the NE system in memory retrieval remains unclear. We developed a chemogenetic activation system based on insect olfactory ionotropic receptors and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons enhanced the retrieval of conditioned taste aversion, which was mediated, in part, through adrenoceptors in the basolateral amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC–amygdala pathway plays an important role in the recall of taste associative memory.

The ability to retrieve memory store in response to the environment is essential for animal behavioral adaptation. Norepinephrine (NE)-containing neurons in the brain play a key role in the modulation of synaptic plasticity underlying various processes of memory formation. However, the role of the central NE system in memory retrieval remains unclear. In this study, we developed a neural chemogenetic activation strategy using insect olfactory Ionotropic Receptors (IRs), and used it for selective stimulation of NE neurons in the locus coeruleus (LC) in transgenic mice. Ligand-induced activation of LC NE neurons resulted in enhancement of the retrieval process of conditioned taste aversion, which was mediated through at least partly adrenergic receptors in the amygdala. Pharmacological blockade of LC activity confirmed the facilitative role of these neurons in memory retrieval. Our findings indicate that the LC-amygdalar pathway is required and sufficient for enhancing the recall of taste associative memory.

Orexin, a hypothalamic neuropeptide, has been revealed to be involved in sleep regulation. To elucidate functions of orexin in brainstem sleep regulation mechanism, we examined the effects of orexin applied from micropipettes with air pressure on neurones in and around the laterodorsal tegmental nucleus (LDT). In five of seven cholinergic neurones and six of nine non‐cholinergic neurones orexin induced long‐lasting excitation. These results suggest that hypothalamic orexin neurones may affect the LDT neurones directly, and thereby participate in control of sleep.

In urethane‐anaesthetized rats, single neuronal activity was recorded extracellularily in and around the laterodorsal tegmental nucleus, and the response to prolactin applied by pressure injection was examined. Three of six cholinergic neurons tested were excited by prolactin for more than 1 min. In two of three non‐cholinergic neurons tested, prolactin application caused excitation which lasted a shorter time than that in the cholinergic neurons. The results suggest that prolactin may have physiological influences on these mesopontine neurons to increase paradoxical sleep.