Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Catecholaminergic cell type-specific expression of Cre recombinase in knock-in transgenic rats generated by the Combi-CRISPR technology.

BACKGROUND: Cell groups containing catecholamines provide a useful model to study the molecular and cellular mechanisms underlying the morphogenesis, physiology, and pathology of the central nervous system. For this purpose, it is necessary to establish a system to induce catecholaminergic group-specific expression of Cre recombinase. Recently, we introduced a gene cassette encoding 2A peptide fused to Cre recombinase into the site between the C-terminus and translational termination codons of the rat tyrosine hydroxylase (TH) open reading frame by the Combi-CRISPR technology, which is a genomic editing method to enable an efficient knock-in (KI) of long DNA sequence into a target site. However, the expression patterns of the transgene and its function as well as the effect of the mutation on the biochemical and behavioral phenotypes in the KI strains have not been characterized yet. NEW METHOD: We aimed to evaluate the usefulness of TH-Cre KI rats as an experimental model for investigating the structure and function of catecholaminergic neurons in the brain. RESULTS: We detected cell type-specific expression of Cre recombinase and site-specific recombination activity in the representative catecholaminergic groups in the TH-Cre KI rat strains. In addition, we measured TH protein levels and catecholamine accumulation in the brain regions, as well as motor, reward-related, and anxiety-like behaviors, indicating that catecholamine metabolism and general behavior are apparently normal in these KI rats. CONCLUSIONS: TH-Cre KI rat strains produced by the Combi-CRISPR system offer a beneficial model to study the molecular and cellular mechanics for the morphogenesis, physiology, and pathology of catecholamine-containing neurons in the brain.

Multimodal Functional Analysis Platform: 1. Ultrathin Fluorescence Endoscope Imaging System Enables Flexible Functional Brain Imaging.

To elucidate the expression mechanisms of brain functions, we have developed an ultrathin fluorescence endoscope imaging system (U-FEIS) that can image cells in the brain at any depth while minimizing the invasion. The endoscope part of U-FEIS consists of a GRIN lens and a 10,000-pixel image fiber with a diameter of 450 µm. The specialized microscope of U-FEIS is within 30 cm square and includes lenses and optical filters optimized for the endoscope. Using U-FEIS, we successfully visualized neurons expressing GFP with single-cell resolution and recorded the multineuronal activities in vitro and in vivo. U-FEIS can also perform imaging and optical stimulation simultaneously. Therefore, U-FEIS should be a powerful optical tool in neuroscience research.

Enhanced Retrieval of Taste Associative Memory by Chemogenetic Activation of Locus Coeruleus Norepinephrine Neurons.

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 melanogaster IR84a 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 and in vivo electrophysiological 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 ß-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 STATEMENT Norepinephrine (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.

Maternal dietary imbalance between omega-6 and omega-3 fatty acids triggers the offspring's overeating in mice.

The increasing prevalence of obesity and its effects on our society warrant intensifying basic animal research for understanding why habitual intake of highly palatable foods has increased due to recent global environmental changes. Here, we report that pregnant mice that consume a diet high in omega-6 (n-6) polyunsaturated fatty acids (PUFAs) and low in omega-3 (n-3) PUFAs (an n-6high/n-3low diet), whose n-6/n-3 ratio is approximately 120, induces hedonic consumption in the offspring by upregulating the midbrain dopaminergic system. We found that exposure to the n-6high/n-3low diet specifically increases the consumption of palatable foods via increased mesolimbic dopamine release. In addition, neurodevelopmental analyses revealed that this induced hedonic consumption is programmed during embryogenesis, as dopaminergic neurogenesis is increased during in utero access to the n-6high/n-3low diet. Our findings reveal that maternal consumption of PUFAs can have long-lasting effects on the offspring's pattern for consuming highly palatable foods.

Sustaining temporal attention prevents habit expression during operant learning in rats.

As repeated operant performance promotes the transition from goal-directed action to habitual response, it appears that action-outcome contingency learning precedes and is necessary for the transition. Meanwhile, it is known that operant performance under a fixed interval (FI) schedule, in which the timing of reinforcement is predictable, is resistant to habit. However, the reason why the FI schedule prevents habit expression remains unclear. We reasoned that sustained attention for monitoring a certain interval might require a goal-directed process and prevent the transition. To verify this hypothesis, rats underwent FI schedule operant training while auditory cues were provided in a manner either contingent or non-contingent with the timing of lever pressing to obtain a reward. The subjects developed a habit with contingent cues, but not with either non-contingent cues or no cues. Overall, we conclude that the release from sustained attentional burden allows the expression of habit. (147 words).

Store-Operated Calcium Channels Are Involved in Spontaneous Slow Calcium Oscillations in Striatal Neurons.

The striatum plays an important role in linking cortical activity to basal ganglia output. Striatal neurons exhibit spontaneous slow Ca2+ oscillations that result from Ca2+ release from the endoplasmic reticulum (ER) induced by the mGluR5-IP3R signaling cascade. The maximum duration of a single oscillatory event is about 300 s. A major question arises as to how such a long-duration Ca2+ elevation is maintained. Store-operated calcium channels (SOCCs) are one of the calcium (Ca2+)-permeable ion channels. SOCCs are opened by activating the metabotropic glutamate receptor type 5 and inositol 1,4,5-trisphosphate receptor (mGluR5-IP3R) signal transduction cascade and are related to the pathophysiology of several neurological disorders. However, the functions of SOCCs in striatal neurons remain unclear. Here, we show that SOCCs exert a functional role in striatal GABAergic neurons. Depletion of calcium stores from the ER induced large, sustained calcium entry that was blocked by SKF96365, an inhibitor of SOCCs. Moreover, the application of SKF96365 greatly reduced the frequency of slow Ca2+ oscillations. The present results indicate that SOCCs contribute to Ca2+ signaling in striatal GABAergic neurons, including medium spiny projection neurons (MSNs) and GABAergic interneurons, through elevated Ca2+ due to spontaneous slow Ca2+ oscillations.

Ethological and multi-behavioral analysis of learning and memory performance in laboratory rodent models.

Behavioral studies using animal models have widely contributed to advancing our understanding of the neuroregulatory mechanisms of human cognitive states and disorders. A variety of behavioral tests and theoretical models have been developed that provide a standardized toolbox of behavioral test paradigms available to researchers, and thus allow rapid progress in studies of the molecular-genetic bases of behavior relevant to neurocognitive diseases. However, a growing effort to utilize standardized paradigms has overlooked the diverse behavioral characteristics of test rodents expressed in standardized test situations. This review describes two popular test paradigms for cognitive assessment in rodents: social recognition and fear conditioning tasks. An extensive assessment of observed behavior during testing indicates a need to further elucidate the sequential strategic processes employed by test animals in conjunction with the use of standardized test settings and dependent variables. The present study calls specific attention to the considerable but improvable problem of the appropriateness and applicability of these standardized test paradigms; it thereby unravels the essential contribution of multi-behavioral assessment to further advancing neuroscience research using rodent behavioral models.

Identification of an unconventional process of instrumental learning characteristically initiated with outcome devaluation-insensitivity and generalized action selection.

The distinction between goal-directed action and habitual response, particularly with respect to moderate or extended appetitive instrumental training, is well documented; however, the propensity toward instrumental behavior in the early training stage has not been elucidated. In this study, we trained Sprague Dawley rats to press a lever to obtain food as an outcome for various time periods and monitored the changes in their sensitivity to outcome devaluation and choice between the levers they had been trained with and unfamiliar levers. After the extensive training with a random interval schedule, the rats were insensitive to outcome devaluation, and exhibited a typical habit-like phenotype, as previously reported, and the untrained leverpresses were relatively rare and sporadic. During the initial stage of training (≤1 week), the rats exhibited a similar insensitivity to the devaluation; however, in contrast to the overtrained condition, they performed distinctive unbiased leverpresses on both the trained and untrained levers. Thus, we propose a possibility that, contrary to the authentic concept that instrumental learning is initiated with an outcome devaluation-sensitive goal-directed stage, under some conditions, this learning can unconventionally begin with the initial stage that is distinct from both goal-directed action and habitual response.

Pre-stress performance in an instrumental training predicts post-stress behavioral alterations in chronically stressed rats.

Stress is a major factor in the development of major depressive disorder (MDD), but few studies have assessed individual risk based on pre-stress behavioral and cognitive traits. To address this issue, we employed appetitive instrumental lever pressing with a progressive ratio (PR) schedule to assess these traits in experimentally naïve Sprague-Dawley rats. Based on four distinct traits that were identified by hierarchical cluster analysis, the animals were classified into the corresponding four subgroups (Low Motivation, Quick Learner, Slow Learner, and Hypermotivation), and exposed to chronic unpredictable stress (CUS) before monitoring their post-stress responses for 4 weeks. The four subgroups represented the following distinct behavioral phenotypes after CUS: the Low Motivation subgroup demonstrated weight loss and a late-developing paradoxical enhancement in PR performance that may be related to inappropriate decision-making in human MDD. The Quick Learner subgroup exhibited a transient loss of motivation and the habituation of serum corticosterone (CORT) response to repeated stress. The Slow Learner subgroup displayed resistance to demotivation and a suppressed CORT response to acute stress. Finally, the Hypermotivation subgroup exhibited resistance to weight loss, habituated CORT response to an acute stress, and a long-lasting amotivation. Overall, we identified causal relationships between pre-stress traits in the performance of the instrumental training and post-stress phenotypes in each subgroup. In addition, many of the CUS-induced phenotypes in rats corresponded to or had putative relationships with representative symptoms in human MDD. We concluded that the consequences of stress may be predictable before stress exposure by determining the pre-stress behavioral or cognitive traits of each individual in rats.

Repeated exposure of adult rats to transient oxidative stress induces various long-lasting alterations in cognitive and behavioral functions.

Exposure of neonates to oxidative stress may increase the risk of psychiatric disorders such as schizophrenia in adulthood. However, the effects of moderate oxidative stress on the adult brain are not completely understood. To address this issue, we systemically administrated 2-cyclohexen-1-one (CHX) to adult rats to transiently reduce glutathione levels. Repeated administration of CHX did not affect the acquisition or motivation of an appetitive instrumental behavior (lever pressing) rewarded by a food outcome under a progressive ratio schedule. In addition, response discrimination and reversal learning were not affected. However, acute CHX administration blunted the sensitivity of the instrumental performance to outcome devaluation, and this effect was prolonged in rats with a history of repeated CHX exposure, representing pro-depression-like phenotypes. On the other hand, repeated CHX administration reduced immobility in forced swimming tests and blunted acute cocaine-induced behaviors, implicating antidepressant-like effects. Multivariate analyses segregated a characteristic group of behavioral variables influenced by repeated CHX administration. Taken together, these findings suggest that repeated administration of CHX to adult rats did not cause a specific mental disorder, but it induced long-term alterations in behavioral and cognitive functions, possibly related to specific neural correlates.

Effects of extended context discrimination training and context extinction on transfer of context dependency of conditioned flavor aversion.

We trained rats in a context discrimination paradigm by pairing a sucrose solution with lithium chloride in one context (conditioning context) and simple exposure to the same fluid in a second (neutral) context to establish a context-dependent aversion to the conditioned fluid. We then investigated whether transfer of the context dependency to a test fluid (a sodium chloride solution) was affected by two post-discrimination training treatments, an extended context discrimination training, and non-reinforced exposure to the conditioning context (context extinction). We found that the context-dependent flavor aversion that had been specific to sucrose transferred to the test fluid after the extensive training (Experiment 1). Context extinction eliminated the transfer effect that had been observed immediately after the context discrimination training (Experiment 2). In addition, an aversion acquired by sucrose through a simple conditioning of sucrose-LiCl pairings did not generalize to the test fluid (Experiment 3). These results emphasize the importance of a Pavlovian excitatory association between the conditioning context and nausea as a primary source of transfer of the context dependency, rather than a generalization of aversion acquired by the conditioned fluid to the test fluid.

Instrumental outcome devaluation with representation-mediated conditioning.

In three experiments, rats were trained to perform two instrumental behaviours (R1 and R2) in the presence of discriminative stimuli (Sd1 and Sd2, respectively) to obtain a common food outcome (O1). Acquisition of the two discriminations was followed by switching the outcome accompanying R2 performance from O1 to a new one (O2). Experiment 1 showed paired presentations of O2 with a lithium chloride (LiCl) injection resulted in a reduction in the R2 performance. In the subsequent two experiments, each Sd was paired with LiCl injection and its effects on outcome consumption and instrumental performance were investigated. A reduction in the O2 consumption subsequent to the Sd devaluation was found in Experiments 2 and 3. Experiment 3 revealed a reduced R2 performance in an extinction test, following the animals' consummatory access to the outcomes in training context. These results demonstrate representation-mediated outcome devaluation in the course of the Sd devaluation.

Reduction of instrumental discrimination performance by post-conditioning devaluation of discriminative stimulus: the effects of novelty in reinforcing outcome and extended training.

In three experiments the effects of post-conditioning pairings of a discriminative stimulus (Sd) with an illness-inducing agent (lithium chloride, LiCl) on subsequent discrimination performance in extinction and consumption of reinforcing outcome were investigated. Rats were trained to choose a correct lever to obtain food pellets, with a light presented on a bulb just above the correct lever serving for the Sd on each trial. After achievement of a criterion of the discrimination, animals received paired or unpaired presentations of the Sds and LiCl injection. In Experiment 1, in which a familiar outcome was given throughout the discrimination training, Sd-LiCl pairings did not reduce either lever-press performance during presentation of the Sds or amount of consumption of outcomes. On the other hand, in Experiment 2 where a novel outcome was introduced in the final two sessions of the discrimination training, subsequent Sd devaluation reduced lever-press performance during presentations of the Sds. Similar findings were obtained in Experiment 3, in which animals were given extended discrimination training with introduction of novel outcomes in the final two sessions. These findings suggest that a representation of the outcome, evoked by presentation of the Sd, and illness were associated in the course of Sd-LiCl pairings but only when a novel outcome was used.