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.

The distribution of Cdh20 mRNA demarcates somatotopic subregions and subpopulations of spiny projection neurons in the rat dorsolateral striatum.

The dorsolateral striatum (DLS) of rodents is functionally subdivided into somatotopic subregions that represent each body part along both the dorsoventral and anteroposterior (A-P) axes and play crucial roles in sensorimotor functions via corticostriatal pathways. However, little is known about the spatial gene expression patterns and heterogeneity of spiny projection neurons (SPNs) within somatotopic subregions. Here, we show that the cell adhesion molecule gene Cdh20, which encodes a Type II cadherin, is expressed in discrete subregions covering the inner orofacial area and part of the forelimb area in the ventral domain of the DLS (v-DLS) in rats. Cdh20-expressing cells were localized in the v-DLS at the intermediate level of the striatum along the A-P axis and could be classified as direct-pathway SPNs or indirect-pathway SPNs. Unexpectedly, comprehensive analysis revealed that Cdh20 is expressed in SPNs in the rat DLS but not in the mouse DLS or the ferret putamen (Pu). Our observations reveal that Cdh20 expression demarcates somatotopic subregions and subpopulations of SPNs specifically in the rat DLS and suggest divergent regulation of genes differentially expressed in the v-DLS and Pu among mammals.

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.

Action Selection and Flexible Switching Controlled by the Intralaminar Thalamic Neurons.

Learning processes contributing to appropriate selection and flexible switching of behaviors are mediated through the dorsal striatum, a key structure of the basal ganglia circuit. The major inputs to striatal subdivisions are provided from the intralaminar thalamic nuclei, including the central lateral nucleus (CL) and parafascicular nucleus (PF). Thalamostriatal neurons in the PF modulate the acquisition and performance of stimulus-response learning. Here, we address the roles of the CL thalamostriatal neurons in learning processes by using a selective neural pathway targeting technique. We show that the CL neurons are essential for the performance of stimulus-response learning and for behavioral flexibility, including reversal and attentional set-shifting of learned responses. In addition, chemogenetic suppression of neural activity supports the requirements of these neurons for behavioral flexibility. Our results suggest that the main contribution of the CL thalamostriatal neurons is functional control of the basal ganglia circuit linked to the prefrontal cortex.

In Vivo Spiking Dynamics of Intra- and Extratelencephalic Projection Neurons in Rat Motor Cortex.

In motor cortex, 2 types of deep layer pyramidal cells send their axons to other areas: intratelencephalic (IT)-type neurons specifically project bilaterally to the cerebral cortex and striatum, whereas neurons of the extratelencephalic (ET)-type, termed conventionally pyramidal tract-type, project ipsilaterally to the thalamus and other areas. Although they have totally different synaptic and membrane potential properties in vitro, little is known about the differences between them in ongoing spiking dynamics in vivo. We identified IT-type and ET-type neurons, as well as fast-spiking-type interneurons, using novel multineuronal analysis based on optogenetically evoked spike collision along their axons in behaving/resting rats expressing channelrhodopsin-2 (Multi-Linc method). We found "postspike suppression" (~100 ms) as a characteristic of ET-type neurons in spike auto-correlograms, and it remained constant independent of behavioral conditions in functionally different ET-type neurons. Postspike suppression followed even solitary spikes, and spike bursts significantly extended its duration. We also observed relatively strong spike synchrony in pairs containing IT-type neurons. Thus, spiking dynamics in IT-type and ET-type neurons may be optimized differently for precise and coordinated motor control.

Enhanced flexibility of place discrimination learning by targeting striatal cholinergic interneurons.

Behavioural flexibility is mediated through the neural circuitry linking the prefrontal cortex and basal ganglia. Here we conduct selective elimination of striatal cholinergic interneurons in transgenic rats by immunotoxin-mediated cell targeting. Elimination of cholinergic interneurons from the dorsomedial striatum (DMS), but not from the dorsolateral striatum, results in enhanced reversal and extinction learning, sparing the acquisition of place discrimination. This enhancement is prevented by infusion of a non-selective muscarinic acetylcholine receptor agonist into the DMS either in the acquisition, reversal or extinction phase. In addition, gene-specific silencing of M4 muscarinic receptor by lentiviral expression of short hairpin RNA (shRNA) mimics the place reversal learning promoted by cholinergic elimination, whereas shRNA-mediated gene silencing of M1 muscarinic receptor shows the normal performance of reversal learning. Our data indicate that DMS cholinergic interneurons inhibit behavioural flexibility, mainly through the M4 muscarinic receptor, suggesting that this role is engaged to the stabilization of acquired reward contingency and the suppression of response switch to changed contingency.

Methylglyoxal (MG) and cerebro-renal interaction: does long-term orally administered MG cause cognitive impairment in normal Sprague-Dawley rats?

Methylglyoxal (MG), one of the uremic toxins, is a highly reactive alpha-dicarbonyl compound. Recent clinical studies have demonstrated the close associations of cognitive impairment (CI) with plasma MG levels and presence of kidney dysfunction. Therefore, the present study aims to examine whether MG is a direct causative substance for CI development. Eight-week-old male Sprague-Dawley (SD) rats were divided into two groups: control (n = 9) and MG group (n = 10; 0.5% MG in drinking water), and fed a normal diet for 12 months. Cognitive function was evaluated by two behavioral tests (object exploration test and radial-arm maze test) in early (4-6 months of age) and late phase (7-12 months of age). Serum MG was significantly elevated in the MG group (495.8 ± 38.1 vs. 244.8 ± 28.2 nM; p < 0.001) at the end of study. The groups did not differ in cognitive function during the course of study. No time-course differences were found in oxidative stress markers between the two groups, while, antioxidants such as glutathione peroxidase and superoxide dismutase activities were significantly increased in the MG group compared to the control. Long-term MG administration to rats with normal kidney function did not cause CI. A counter-balanced activation of the systemic anti-oxidant system may offset the toxicity of MG in this model. Pathogenetic significance of MG for CI requires further investigation.

Neural circuit mechanism for learning dependent on dopamine transmission: roles of striatal direct and indirect pathways in sensory discrimination.

The dorsal striatum in basal ganglia circuit mediates learning processes contributing to instrumental motor actions. The striatum receives excitatory inputs from many cortical areas and the thalamic nuclei and dopaminergic inputs from the ventral midbrain and projects to the output nuclei through direct and indirect pathways. The neural mechanism remains unclear as to how these striatofugal pathways control the learning processes of instrumental actions. Here, we addressed the behavioral roles of the two striatofugal pathways in the performance of sensory discrimination by using immunotoxin (IT)-mediated cell targeting. IT targeting of the striatal direct pathway in mutant mice lengthened the response time but did not affect the accuracy of the response selection in visual discrimination. Subregion-specific pathway targeting revealed a delay in motor responses generated by elimination of the direct pathway arising from the dorsomedial striatum (DMS) but not from the dorsolateral striatum (DLS). These findings indicate that the direct pathway, in particular that from the DMS, contributes to the regulation of the response time in visual discrimination. In addition, IT targeting of the striatal indirect pathway originating from the DLS in transgenic rats impaired the accuracy of response selection in auditory discrimination, whereas the response time remained normal. These data demonstrate that the DLS-derived indirect pathway plays an essential role in the control of the selection accuracy of learned motor responses. Our results suggest that striatal direct and indirect pathways act cooperatively to regulate the selection accuracy and response time of learned motor actions in the performance of discriminative learning.

The ubiquitin ligase Praja1 reduces NRAGE expression and inhibits neuronal differentiation of PC12 cells.

Evidence suggests that regulated ubiquitination of proteins plays a critical role in the development and plasticity of the central nervous system. We have previously identified the ubiquitin ligase Praja1 as a gene product induced during fear memory consolidation. However, the neuronal function of this enzyme still needs to be clarified. Here, we investigate its involvement in the nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells. Praja1 co-localizes with cytoskeleton components and the neurotrophin receptor interacting MAGE homologue (NRAGE). We observed an enhanced expression of Praja1 after 3 days of NGF treatment and a suppression of neurite formation upon Praja1 overexpression in stably transfected PC12 cell lines, which was associated with a proteasome-dependent reduction of NRAGE levels. Our data suggest that Praja1, through ubiquitination and degradation of NRAGE, inhibits neuronal differentiation. The two murine isoforms, Praja1.1 and Praja1.2, appear to be functionally homologous in this respect.

Protocadherin 17 regulates presynaptic assembly in topographic corticobasal Ganglia circuits.

Highly topographic organization of neural circuits exists for the regulation of various brain functions in corticobasal ganglia circuits. Although neural circuit-specific refinement during synapse development is essential for the execution of particular neural functions, the molecular and cellular mechanisms for synapse refinement are largely unknown. Here, we show that protocadherin 17 (PCDH17), one of the nonclustered δ2-protocadherin family members, is enriched along corticobasal ganglia synapses in a zone-specific manner during synaptogenesis and regulates presynaptic assembly in these synapses. PCDH17 deficiency in mice causes facilitated presynaptic vesicle accumulation and enhanced synaptic transmission efficacy in corticobasal ganglia circuits. Furthermore, PCDH17(-/-) mice exhibit antidepressant-like phenotypes that are known to be regulated by corticobasal ganglia circuits. Our findings demonstrate a critical role for PCDH17 in the synaptic development of specific corticobasal ganglia circuits and suggest the involvement of PCDH17 in such circuits in depressive behaviors.

Striatal indirect pathway contributes to selection accuracy of learned motor actions.

The dorsal striatum, which contains the dorsolateral striatum (DLS) and dorsomedial striatum (DMS), integrates the acquisition and implementation of instrumental learning in cooperation with the nucleus accumbens (NAc). The dorsal striatum regulates the basal ganglia circuitry through direct and indirect pathways. The mechanism by which these pathways mediate the learning processes of instrumental actions remains unclear. We investigated how the striatal indirect (striatopallidal) pathway arising from the DLS contributes to the performance of conditional discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D(2) receptor in the DLS of transgenic rats resulted in selective, efficient elimination of the striatopallidal pathway. This elimination impaired the accuracy of response selection in a two-choice reaction time task dependent on different auditory stimuli. The impaired response selection was elicited early in the test sessions and was gradually restored as the sessions continued. The restoration from the deficits in auditory discrimination was prevented by excitotoxic lesion of the NAc but not by that of the DMS. In addition, lesion of the DLS mimicked the behavioral consequence of the striatopallidal removal at the early stage of test sessions of discriminative performance. Our results demonstrate that the DLS-derived striatopallidal pathway plays an essential role in the execution of conditional discrimination, showing its contribution to the control of selection accuracy of learned motor responses. The results also suggest the presence of a mechanism that compensates for the learning deficits during the repetitive sessions, at least partly, demanding accumbal function.

Striatal direct pathway modulates response time in execution of visual discrimination.

The dorsal striatum in the basal ganglia circuitry is a principal structure that mediates the acquisition and performance of instrumental learning. The projections from the dorsal striatum are composed of two subpopulations of medium spiny neurons that constitute the direct and indirect pathways. The mechanism by which these striatal projections control the learning processes of instrumental actions remains unknown. We addressed the behavioral role of the striatal direct (striatonigral) pathway in the performance of visual discrimination. Immunotoxin targeting of the striatal neuronal type containing dopamine D(1) receptor in mice resulted in a moderate level of elimination of the striatonigral pathway. Targeting of the neural pathway from the whole region of the dorsal striatum lengthened the response time but did not affect the accuracy of response selection in a two-choice reaction time task dependent on light stimulus. This lengthened motor response was induced early in the test sessions and was gradually restored to normal levels during repetitive sessions. In addition, subregion-specific pathway targeting revealed that the delay in learned motor response was generated by the elimination of the striatonigral pathway arising from the dorsomedial striatum but not from the dorsolateral striatum. Our findings indicate that the striatonigral pathway, in particular from the dorsomedial striatum, contributes to the regulation of response time in the execution of visual discrimination. The restoration of motor response deficits during repetitive sessions suggests the presence of a mechanism by which the response facilitation is acquired through continuation of learning despite the removal of the striatonigral pathway.

Selective neural pathway targeting reveals key roles of thalamostriatal projection in the control of visual discrimination.

The dorsal striatum receives converging excitatory inputs from diverse brain regions, including the cerebral cortex and the intralaminar/midline thalamic nuclei, and mediates learning processes contributing to instrumental motor actions. However, the roles of each striatal input pathway in these learning processes remain uncertain. We developed a novel strategy to target specific neural pathways and applied this strategy for studying behavioral roles of the pathway originating from the parafascicular nucleus (PF) and projecting to the dorsolateral striatum. A highly efficient retrograde gene transfer vector encoding the recombinant immunotoxin (IT) receptor was injected into the dorsolateral striatum in mice to express the receptor in neurons innervating the striatum. IT treatment into the PF of the vector-injected animals caused a selective elimination of neurons of the PF-derived thalamostriatal pathway. The elimination of this pathway impaired the response selection accuracy and delayed the motor response in the acquisition of a visual cue-dependent discrimination task. When the pathway elimination was induced after learning acquisition, it disturbed the response accuracy in the task performance with no apparent change in the response time. The elimination did not influence spontaneous locomotion, methamphetamine-induced hyperactivity, and motor skill learning that demand the function of the dorsal striatum. These results demonstrate that thalamostriatal projection derived from the PF plays essential roles in the acquisition and execution of discrimination learning in response to sensory stimulus. The temporal difference in the pathway requirement for visual discrimination suggests a stage-specific role of thalamostriatal pathway in the modulation of response time of learned motor actions.

[Functional analysis of the roles of direct and indirect pathways by using immunotoxin-mediated cell targeting approach].

The neural circuit connecting the cerebral cortex and the basal ganglia mediates a variety of brain functions including voluntary movement, motor learning, and reinforcement learning. These functions are dependent on midbrain dopamine systems that innervate the prefrontal cortex and the striatum. The pathogenesis of certain neurological and neuropsychiatric diseases involves the dysfunction of these dopamin systems; some of these diseases include Parkinson's disease, schizophrenia, and attention deficit hyperactivity disorder. To understand the physiology and pathology of brain functions, the mechanisms of neural circuitry that controls behaviors should be studied. Immunotoxin-mediated cell targeting is an approach employed in transgenic animals to eliminated specific neuronal types from a neuronal circuitry. This approach has been used to study the neural circuit mechanism in the central and peripheral nervous systems. Here, we describe the use of immunotoxin-mediated cell targeting for studying the neural circuitry that underlies the motor behavior demonstrated in response to systemic dopamine stimulation; further, we propose the potential mechanism that controls direct and indirect striatal pathway-dependent behavior.