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.

RhoA downregulation in the murine intestinal epithelium results in chronic Wnt activation and increased tumorigenesis.

Rho GTPases are molecular switches regulating multiple cellular processes. To investigate the role of RhoA in normal intestinal physiology, we used a conditional mouse model overexpressing a dominant negative RhoA mutant (RhoAT19N) in the intestinal epithelium. Although RhoA inhibition did not cause an overt phenotype, increased levels of nuclear ß-catenin were observed in the small intestinal epithelium of RhoAT19N mice, and the overexpression of multiple Wnt target genes revealed a chronic activation of Wnt signaling. Elevated Wnt signaling in RhoAT19N mice and intestinal organoids did not affect the proliferation of intestinal epithelial cells but significantly interfered with their differentiation. Importantly, 17-month-old RhoAT19N mice showed a significant increase in the number of spontaneous intestinal tumors. Altogether, our results indicate that RhoA regulates the differentiation of intestinal epithelial cells and inhibits tumor initiation, likely through the control of Wnt signaling, a key regulator of proliferation and differentiation in the intestine.

Noninvasive imaging of rat-derived microglia and its reactivity to inflammatory molecules via acoustic impedance microscopy.

PURPOSE: Microglia, the brain's immune cells, play important roles in neuronal differentiation, survival, and death. The function of microglia is deeply related to the morphologies; however, it is too complex to observe conventionally and identify the condition of living microglia using optical microscopes. Herein, we proposed a new method to observe living cultured microglia and their reactivity to inflammation via the acoustic impedance mode of a scanning acoustic microscope. METHODS: Primary cultured microglia collected from rat pups exposed to acetamiprid, an insecticide, in utero were observed with both acoustic interface impedance mode (C-mode) and transparent three-dimensional impedance mode (B-mode). RESULTS: We characterized microglia into four types based on the results obtained from acoustic impedance, cytoskeletal information, and laser confocal imaging. Biphasic acoustic observation using B-mode and C-mode gave us information regarding the dynamic morphologies of living microglia treated with adenosine triphosphate (ATP) (600 µmol/L), which reflects distress signals from inflamed neurons. Acetamiprid exposure induced microglia response even in the neonatal period. ATP stimulus altered the shape and thickness of microglia with a change in the bulk modulus of the cell. Three-dimensional alteration with ATP stimulus could be observed only after biphasic acoustic observation using B-mode and C-mode. This acoustic observation was consistent with confocal observation using anti-Iba-1 and P2Y12 immunocytochemistry. CONCLUSION: This study demonstrated the adequacy of using a scanning acoustic microscope in analyzing microglia's shape, motility, and response to inflammation.

Two parabrachial Cck neurons involved in the feedback control of thirst or salt appetite.

Thirst and salt appetite are temporarily suppressed after water and salt ingestion, respectively, before absorption; however, the underlying neural mechanisms remain unclear. The parabrachial nucleus (PBN) is the relay center of ingestion signals from the digestive organs. We herein identify two distinct neuronal populations expressing cholecystokinin (Cck) mRNA in the lateral PBN that are activated in response to water and salt intake, respectively. The two Cck neurons in the dorsal-lateral compartment of the PBN project to the median preoptic nucleus and ventral part of the bed nucleus of the stria terminalis, respectively. The optogenetic stimulation of respective Cck neurons suppresses thirst or salt appetite under water- or salt-depleted conditions. The combination of optogenetics and in vivo Ca2+ imaging during ingestion reveals that both Cck neurons control GABAergic neurons in their target nuclei. These findings provide the feedback mechanisms for the suppression of thirst and salt appetite after ingestion.

Protocol for highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence in rodents.

We present a protocol to induce Cre-dependent transgene expression in specific cell types in the rat brain, suppressing a leak expression in off-target cells, by using a flip-excision switch system with a unilateral spacer sequence. We describe steps for construction of transfer plasmids, preparation of adeno-associated viral vectors, intracranial injection, and detection of transgene expression. Our protocol provides a useful strategy for a better understanding of the structure and function of specific cell types in the complex neural circuit. For complete details on the use and execution of this protocol, please refer to Matsushita et al.1.

An intact pituitary vasopressin system is critical for building a robust circadian clock in the suprachiasmatic nucleus.

The circadian clock is a biological timekeeping system that oscillates with a circa-24-h period, reset by environmental timing cues, especially light, to the 24-h day-night cycle. In mammals, a "central" clock in the hypothalamic suprachiasmatic nucleus (SCN) synchronizes "peripheral" clocks throughout the body to regulate behavior, metabolism, and physiology. A key feature of the clock's oscillation is resistance to abrupt perturbations, but the mechanisms underlying such robustness are not well understood. Here, we probe clock robustness to unexpected photic perturbation by measuring the speed of reentrainment of the murine locomotor rhythm after an abrupt advance of the light-dark cycle. Using an intersectional genetic approach, we implicate a critical role for arginine vasopressin pathways, both central within the SCN and peripheral from the anterior pituitary.

Anatomical identification of a corticocortical top-down recipient inhibitory circuitry by enhancer-restricted transsynaptic tracing.

Despite the importance of postsynaptic inhibitory circuitry targeted by mid/long-range projections (e.g., top-down projections) in cognitive functions, its anatomical properties, such as laminar profile and neuron type, are poorly understood owing to the lack of efficient tracing methods. To this end, we developed a method that combines conventional adeno-associated virus (AAV)-mediated transsynaptic tracing with a distal-less homeobox (Dlx) enhancer-restricted expression system to label postsynaptic inhibitory neurons. We called this method "Dlx enhancer-restricted Interneuron-SpECific transsynaptic Tracing" (DISECT). We applied DISECT to a top-down corticocortical circuit from the secondary motor cortex (M2) to the primary somatosensory cortex (S1) in wild-type mice. First, we injected AAV1-Cre into the M2, which enabled Cre recombinase expression in M2-input recipient S1 neurons. Second, we injected AAV1-hDlx-flex-green fluorescent protein (GFP) into the S1 to transduce GFP into the postsynaptic inhibitory neurons in a Cre-dependent manner. We succeeded in exclusively labeling the recipient inhibitory neurons in the S1. Laminar profile analysis of the neurons labeled via DISECT indicated that the M2-input recipient inhibitory neurons were distributed in the superficial and deep layers of the S1. This laminar distribution was aligned with the laminar density of axons projecting from the M2. We further classified the labeled neuron types using immunohistochemistry and in situ hybridization. This post hoc classification revealed that the dominant top-down M2-input recipient neuron types were somatostatin-expressing neurons in the superficial layers and parvalbumin-expressing neurons in the deep layers. These results demonstrate that DISECT enables the investigation of multiple anatomical properties of the postsynaptic inhibitory circuitry.

Reward expectation enhances action-related activity of nigral dopaminergic and two striatal output pathways.

Neurons comprising nigrostriatal system play important roles in action selection. However, it remains unclear how this system integrates recent outcome information with current action (movement) and outcome (reward or no reward) information to achieve appropriate subsequent action. We examined how neuronal activity of substantia nigra pars compacta (SNc) and dorsal striatum reflects the level of reward expectation from recent outcomes in rats performing a reward-based choice task. Movement-related activity of direct and indirect pathway striatal projection neurons (dSPNs and iSPNs, respectively) were enhanced by reward expectation, similarly to the SNc dopaminergic neurons, in both medial and lateral nigrostriatal projections. Given the classical basal ganglia model wherein dopamine stimulates dSPNs and suppresses iSPNs through distinct dopamine receptors, dopamine might not be the primary driver of iSPN activity increasing following higher reward expectation. In contrast, outcome-related activity was affected by reward expectation in line with the classical model and reinforcement learning theory, suggesting purposive effects of reward expectation.

Dissection of insular cortex layer 5 reveals two sublayers with opposing modulatory roles in appetitive drinking behavior.

The insular cortex (insula) is known to play a modulatory role in feeding and drinking. Previous studies have revealed anterior-posterior differences of subcortical projections and roles for the insula, yet the anatomical and functional heterogeneity among the cortical layers remains poorly understood. Here, we show that layer 5 of the mouse dysgranular insula has two distinct neuronal subpopulations along the entire anterior-posterior axis: The L5a population, expressing NECAB1, projects bilaterally to the lateral and capsular divisions of the central amygdala, and the L5b population, expressing CTIP2, projects ipsilaterally to the parasubthalamic nucleus and the medial division of the central amygdala. Optogenetically activating L5a and L5b neuronal populations in thirsty male mice led to suppressed and facilitated water spout licking, respectively, without avoidance against or preference for the spout paired with the opto-stimulation. Our results suggest sublayer-specific bidirectional modulatory roles of insula layer 5 in the motivational aspect of appetitive behavior.

Reinstating olfactory bulb-derived limbic gamma oscillations alleviates depression-like behavioral deficits in rodents.

Although the etiology of major depressive disorder remains poorly understood, reduced gamma oscillations is an emerging biomarker. Olfactory bulbectomy, an established model of depression that reduces limbic gamma oscillations, suffers from non-specific effects of structural damage. Here, we show that transient functional suppression of olfactory bulb neurons or their piriform cortex efferents decreased gamma oscillation power in limbic areas and induced depression-like behaviors in rodents. Enhancing transmission of gamma oscillations from olfactory bulb to limbic structures by closed-loop electrical neuromodulation alleviated these behaviors. By contrast, silencing gamma transmission by anti-phase closed-loop stimulation strengthened depression-like behaviors in naive animals. These induced behaviors were neutralized by ketamine treatment that restored limbic gamma power. Taken together, our results reveal a causal link between limbic gamma oscillations and depression-like behaviors in rodents. Interfering with these endogenous rhythms can affect behaviors in rodent models of depression, suggesting that restoring gamma oscillations may alleviate depressive symptoms.

Highly selective transgene expression through the flip-excision switch system by using a unilateral spacer sequence.

The flip-excision switch (FLEX) system with an adeno-associated viral (AAV) vector allows expression of transgenes in specific cell populations having Cre recombinase. A significant issue with this system is non-specific expression of transgenes in tissues after vector injection. We show here that Cre-independent recombination events in the AAV genome carrying the FLEX sequence occur mainly during the production of viral vectors in packaging cells, which results in transgene expression in off-target populations. Introduction of a relatively longer nucleotide sequence between two recognition sites at the unilateral side of the transgene cassette, termed a unilateral spacer sequence (USS), is useful to suppress the recombination in the viral genome, leading to the protection of non-specific transgene expression with enhanced gene expression selectivity. Our FLEX/USS system offers a powerful strategy for highly specific Cre-dependent transgene expression, aiming at various applications for structural and functional analyses of target cell populations.

Involvement of muscarinic receptors in psychomotor hyperactivity in dopamine-deficient mice.

Dopamine-deficient (DD) mice exhibit psychomotor hyperactivity that might be related to a decrease in muscarinic signaling. In the present study, muscarinic acetylcholine receptor M2 (CHRM2) density decreased in the cortex in DD mice. This is significant because cortical CHRM2 acts as an autoreceptor; therefore, changes in CHRM2 levels could alter acetylcholine in DD mice. We also found that the CHRM1/CHRM4 agonist xanomeline and CHRM2 agonist arecaidine propargyl ester tosylate inhibited hyperactivity in DD mice, suggesting that postsynaptic CHRM1 and CHRM2 and presynaptic CHRM2 may be involved in hyperactivity in DD mice.

Cholinergic regulation of object recognition memory.

Object recognition memory refers to a basic memory mechanism to identify and recall various features of objects. This memory has been investigated by numerous studies in human, primates and rodents to elucidate the neuropsychological underpinnings in mammalian memory, as well as provide the diagnosis of dementia in some neurological diseases, such as Alzheimer's disease and Parkinson's disease. Since Alzheimer's disease at the early stage is reported to be accompanied with cholinergic cell loss and impairment in recognition memory, the central cholinergic system has been studied to investigate the neural mechanism underlying recognition memory. Previous studies have suggested an important role of cholinergic neurons in the acquisition of some variants of object recognition memory in rodents. Cholinergic neurons in the medial septum and ventral diagonal band of Broca that project mainly to the hippocampus and parahippocampal area are related to recognition memory for object location. Cholinergic projections from the nucleus basalis magnocellularis innervating the entire cortex are associated with recognition memory for object identification. Especially, the brain regions that receive cholinergic projections, such as the perirhinal cortex and prefrontal cortex, are involved in recognition memory for object-in-place memory and object recency. In addition, experimental studies using rodent models for Alzheimer's disease have reported that neurodegeneration within the central cholinergic system causes a deficit in object recognition memory. Elucidating how various types of object recognition memory are regulated by distinct cholinergic cell groups is necessary to clarify the neuronal mechanism for recognition memory and the development of therapeutic treatments for dementia.

Characterization of the fatty acid profile in the ventral midbrain of mice exposed to dietary imbalance between omega-6 and omega-3 fatty acids during specific life stages.

OBJECTIVE: Omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFAs) are essential nutrients. Dietary imbalance between these PUFAs, in particular high in n-6 PUFAs and low in n-3 PUFAs (n-6high/n-3low), is common in modern society. We have previously reported that C57BL/6 mouse male offspring derived from mothers exposed to an n-6high/n-3low diet during the gestation had an augmented ventral midbrain dopamine system in adulthood; however, the fatty acid composition in this brain region has not yet been investigated. This follow-up study aims to characterize the fatty acid profile of the ventral midbrain of mice exposed to the n-6high/n-3low diet during specific life stages. RESULTS: n-6 PUFAs, especially linoleic acid, were increased in the ventral midbrain of offspring exposed to the n-6high/n-3low diet during the gestation compared to those exposed to a well-balanced control diet throughout life. On the other hand, n-3 PUFAs, especially docosahexaenoic acid, were decreased in the ventral midbrain of offspring exposed to the n-6high/n-3low diet during the gestation, lactation, or postweaning period compared to those exposed to the control diet throughout life. Thus, exposure to the n-6high/n-3low diet in pregnancy increases linoleic acid and that in any life stage decreases docosahexaenoic acid in the offspring's ventral midbrain.

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.

Therapeutic Effects of Quetiapine and 5-HT1A Receptor Agonism on Hyperactivity in Dopamine-Deficient Mice.

Some diseases that are associated with dopamine deficiency are accompanied by psychiatric symptoms, including Parkinson's disease. However, the mechanism by which this occurs has not been clarified. Previous studies found that dopamine-deficient (DD) mice exhibited hyperactivity in a novel environment. This hyperactivity is improved by clozapine and donepezil, which are used to treat psychiatric symptoms associated with dopamine deficiency (PSDD). We considered that DD mice could be used to study PSDD. In the present study, we sought to identify the pharmacological mechanism of PSDD. We conducted locomotor activity tests by administering quetiapine and drugs that have specific actions on serotonin (5-hydroxytryptamine [5-HT]) receptors and muscarinic receptors. Changes in neuronal activity that were induced by drug administration in DD mice were evaluated by examining Fos immunoreactivity. Quetiapine suppressed hyperactivity in DD mice while the 5-HT1A receptor antagonist WAY100635 inhibited this effect. The number of Fos-positive neurons in the median raphe nucleus increased in DD mice that exhibited hyperactivity and was decreased by treatment with quetiapine and 5-HT1A receptor agonists. In conclusion, hyperactivity in DD mice was ameliorated by quetiapine, likely through 5-HT1A receptor activation. These findings suggest that 5-HT1A receptors may play a role in PSDD, and 5-HT1A receptor-targeting drugs may help improve PSDD.

Recording and manipulation of the maternal oxytocin neural activities in mice.

Pulsatile release of the hormone oxytocin (OT) mediates uterine contraction during parturition and milk ejection during lactation.1-3 These pulses are generated by the unique activity patterns of the central neuroendocrine OT neurons located in the paraventricular and supraoptic hypothalamus. Classical studies have characterized putative OT neurons by in vivo extracellular recording techniques in rats and rabbits.1,4-10 Due to technical limitations, however, the identity of OT neurons in these previous studies was speculative based on their electrophysiological characteristics and axonal projection to the posterior pituitary, not on OT gene expression. To pinpoint OT neural activities among other hypothalamic neurons that project to the pituitary11,12 and make better use of cell-type-specific neuroscience toolkits,13 a mouse model needs to be developed for the studies of parturition and lactation. We herein introduce viral genetic approaches in mice to characterize the maternal activities of OT neurons by fiber photometry. A sharp photometric peak of OT neurons appeared at approximately 520 s following simultaneous suckling stimuli from three pups. The amplitude of the peaks increased as the mother mice experienced lactation, irrespective of the age of the pups, suggesting the intrinsic plasticity of maternal OT neurons. Based on a mono-synaptic input map to OT neurons, we pharmacogenetically activated the inhibitory neurons in the bed nucleus of the stria terminalis and found the suppression of the activities of OT neurons. Collectively, our study illuminates temporal dynamics in the maternal neural activities of OT neurons and identifies one of its modulatory inputs.

Fibril matrices created with collagen from the marine fish barramundi for use in conventional three-dimensional cell culture.

Collagen obtained from fish offal (skin, scales, and bones) is required from some religious and ethnic groups, thus indirectly increasing demands for fish collagen for biomedical applications. The limitation of fish collagen is its lower thermal stability compared to mammalian collagen. In this study, we focused on collagen extracted from scales of the marine fish barramundi (Lates calcarifer) and demonstrated the suitability for the collagen to be utilized in collagen fibril matrices (CFM). Collagen was extracted from the scales through pepsin-digestion and purified (designated as "BC"). The denaturation temperature (Td) for BC was determined to be 36.4 °C, one of the highest among fish collagens. BC formed CFM which was thermally stable at 37 °C, while Td was lower than 37 °C. This could be explained by the fast fibril formation, initiating at temperatures near 20 °C in a temperature-elevated process. As a result, the NIH3T3 cells were successfully encapsulated in the CFM of BC and cultured three-dimensionally for 7 d. The cells spread and exhibited well-developed pseudopodia in the CFM of BC as observed in the CFM of pig collagen matrices. This is the first report on fish CFM used for conventional 3-D cell culture.

Vessel Rupture Complication due to Ballooning of the Distal Segment of a JET 7 Xtra Flex Reperfusion Catheter during Angiogram.

Objective: We present a case of intraprocedural device malfunction related to the JET 7 Xtra Flex reperfusion catheter during mechanical thrombectomy. Case Presentation: A 92-year-old man presented with sudden right hemiparesis with a National Institutes of Health Stroke Scale score of 22. His left middle cerebral artery (M1) was occluded, and emergency mechanical thrombectomy was performed. After partial recanalization was achieved, angiography through a JET 7 Xtra Flex was attempted. After manual injection of contrast media via a 10-mL syringe through the JET 7 Xtra Flex, the catheter moved, jumping forward, and the distal tip of the catheter expanded and ruptured. This resulted in intracranial vessel damage and subsequent patient death. Conclusion: Contrast media must not be injected through the JET 7 Xtra Flex. If contrast media needs to be injected for angiography during mechanical thrombectomy with a reperfusion catheter, it should always be through the guide catheter.