Dopaminergic Signaling in the Nucleus Accumbens Modulates Stress-Coping Strategies during Inescapable Stress.

Maladaptation to stress is a critical risk factor in stress-related disorders, such as major depression and post-traumatic stress disorder (PTSD). Dopamine signaling in the nucleus accumbens (NAc) has been shown to modulate behavior by reinforcing learning and evading aversive stimuli, which are important for the survival of animals under environmental challenges such as stress. However, the mechanisms through which dopaminergic transmission responds to stressful events and subsequently regulates its downstream neuronal activity during stress remain unknown. To investigate how dopamine signaling modulates stress-coping behavior, we measured the subsecond fluctuation of extracellular dopamine concentration and pH using fast scanning cyclic voltammetry (FSCV) in the NAc, a postsynaptic target of midbrain dopaminergic neurons, in male mice engaged in a tail suspension test (TST). The results revealed a transient decrease in dopamine concentration and an increase in pH levels when the animals changed behaviors, from being immobile to struggling. Interestingly, optogenetic inhibition of dopamine release in NAc, potentiated the struggling behavior in animals under the TST. We then addressed the causal relationship of such a dopaminergic transmission with behavioral alterations by knocking out both the dopamine receptors, i.e., D1 and D2, in the NAc using viral vector-mediated genome editing. Behavioral analyses revealed that male D1 knock-out mice showed significantly more struggling bouts and longer struggling durations during the TST, while male D2 knock-out mice did not. Our results therefore indicate that D1 dopaminergic signaling in the NAc plays a pivotal role in the modulation of stress-coping behaviors in animals under tail suspension stress.SIGNIFICANCE STATEMENT The tail suspension test (TST) has been widely used as a despair-based behavioral assessment to screen the antidepressant so long. Despite its prevalence in the animal studies, the neural substrate underlying the changes of behavior during the test remains unclear. This study provides an evidence for a role of dopaminergic transmission in the modulation of stress-coping behavior during the TST, a despair test widely used to screen the antidepressants in rodents. Taking into consideration the fact that the dopamine metabolism is upregulated by almost all antidepressants, a part of which acts directly on the dopaminergic transmission, current results would uncover the molecular mechanism through which the dopaminergic signaling mediates antidepressant effect with facilitation of the recovery from the despair-like behavior in the TST.

Electrophysiological and pharmacological characterization of spreading depolarization in the adult zebrafish tectum.

Spreading depolarization (SD) is a slowly propagating wave of neuronal and glial depolarization. A growing number of studies show that SD and SD-like phenomena play a role in neurological disorders such as migraine, stroke, and traumatic brain injury. Despite the clinical importance of SD, its underlying molecular and cellular mechanisms remain elusive, possibly because of insufficient animal model allowing genetic manipulation. Such a model would also allow high-throughput screening for SD-suppressing drug development. To address this, we developed a novel experimental system to study SD using zebrafish. Electrophysiological recordings in the immobilized adult zebrafish revealed that increasing extracellular potassium concentration elicited SD with a large and long-lasting negative shift of direct current (DC) potential in the optic tectum. It also reduced the oscillatory activity in the extracellular field potential and increased the expression of the immediate early gene c-fos. Pharmacological blocking of the N-methyl-d-aspartate (NMDA) glutamate receptor attenuated the propagation of SD, suggesting that glutamatergic neurotransmission mediated tectal SD in zebrafish. Our analyses revealed that the zebrafish tectum and rodent cortex had similar SD kinetics. The current study provides electrophysiological and pharmacological evidence that zebrafish SD and mammal SD are comparable. This zebrafish SD model is suitable for genetic manipulation and cost-effective high-throughput screening. It could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.NEW & NOTEWORTHY Previous studies have implicated spreading depolarization (SD) in stroke and migraine. Here, we demonstrate SD, for the first time, in the adult zebrafish tectum showing waveform kinetics, c-fos expression, and attenuation by N-methyl-d-aspartate glutamate receptor blocker as observed in the rodent cortex. Since the zebrafish is an animal model amenable to genetic manipulation and chemical screening, this result could pave the way to novel diagnostic and therapeutic methods applicable to SD-associated neurological disorders.

Glial glutamate transporter GLT-1 determines susceptibility to spreading depression in the mouse cerebral cortex.

Cortical spreading depression (CSD) is a pathological neural excitation that underlies migraine pathophysiology. Since glutamate receptor antagonists impair CSD propagation, susceptibility to CSD might be determined by any of the neuronal (excitatory amino acid carrier 1 [EAAC1]) and glial (GLutamate ASpartate Transporter [GLAST] and glial glutamate transporter 1 [GLT-1]) glutamate transporters, which are responsible for clearing extracellular glutamate. To investigate this hypothesis, we performed electrophysiological, hemodynamic, and electrochemical analyses using EAAC1- (EAAC1 KO), GLAST- (GLAST KO), and conditional GLT1-1-knockout mice (GLT-1 cKO) to assess altered susceptibility to CSD. Despite the incomplete deletion of the gene in the cerebral cortex, GLT-1 cKO mice exhibited significant reduction of GLT-1 protein in the brain without apparent alteration of the cytoarchitecture in the cerebral cortex. Physiological analysis revealed that GLT-1 cKO showed enhanced susceptibility to CSD elicited by chemical stimulation with increased CSD frequency and velocity compared to GLT-1 control. In contrast, the germ-line EAAC1 and GLAST KOs showed no such effect. Intriguingly, both field potential and cerebral blood flow showed faster dynamics with narrower CSD than the controls. An enzyme-based biosensor revealed more rapid accumulation of glutamate in the extracellular space in GLT-1 cKO mice during the early phase of CSD than in GLT-1 control, resulting in an increased susceptibility to CSD. These results provided the first evidence for a novel role of GLT-1 in determining susceptibility to CSD.

Alternative splicing in the C-terminal tail of Cav2.1 is essential for preventing a neurological disease in mice.

Alternative splicing (AS) that occurs at the final coding exon (exon 47) of the Cav2.1 voltage-gated calcium channel (VGCC) gene produces two major isoforms in the brain, MPI and MPc. These isoforms differ in their splice acceptor sites; human MPI is translated into a polyglutamine tract associated with spinocerebellar ataxia type 6 (SCA6), whereas MPc splices to an immediate stop codon, resulting in a shorter cytoplasmic tail. To gain insight into the functional role of the AS in vivo and whether modulating the splice patterns at this locus can be a potential therapeutic strategy for SCA6, here we created knockin mice that exclusively express MPc by inserting the splice-site mutation. The resultant Cacna1aCtmKO/CtmKO mice developed non-progressive neurological phenotypes, featuring early-onset ataxia and absence seizure without significant alterations in the basic properties of the channel. Interactions of Cav2.1 with Cavß4 and Rimbp2 were significantly reduced while those with GABAB2 were enhanced in the cerebellum of Cacna1aCtmKO/CtmKO mice. Treatment with the GABAB antagonist CGP35348 partially rescued the motor impairments seen in Cacna1aCtmKO/CtmKO mice. These results suggest that the carboxyl-terminal domain of Cav2.1 is not essential for maintaining the basic properties of the channel in the cerebellar Purkinje neurons but is involved in multiple interactions of Cav2.1 with other proteins, and plays an essential role in preventing a complex neurological disease.

Toward an understanding of the habenula's various roles in human depression.

The habenula is an evolutionarily conserved structure in the vertebrate brain. Lesion and electrophysiological studies in animals have suggested that it is involved in the regulation of monoaminergic activity through projection to the brain stem nuclei. Since studies in animal models of depression and human functional imaging have indicated that increased activity of the habenula is associated with depressive phenotypes, this structure has attracted a surge of interest in neuroscience research. According to pathway- and cell-type-specific dissection of habenular function in animals, we have begun to understand how the heterogeneity of the habenula accounts for alteration of diverse physiological functions in depression. Indeed, recent studies have revealed that the subnuclei embedded in the habenula show a wide variety of molecular profiles not only in neurons but also in glial cells implementing the multifaceted regulatory mechanism for output from the habenula. In this review, we overview the known facts on mediolateral subdivision in the habenular structure, then discuss heterogeneity of the habenular structure from the anatomical and functional viewpoint to understand its emerging role in diverse neural functions relevant to depressive phenotypes. Despite the prevalent use of antidepressants acting on monoamine metabolisms, ~30% of patients with major depression are reported to be treatment-resistant. Thus, cellular mechanisms deciphering such diversity in depressive symptoms would be a promising candidate for the development of new antidepressants.

Region-specific deletions of the glutamate transporter GLT1 differentially affect seizure activity and neurodegeneration in mice.

Glial glutamate transporter GLT1 plays a key role in the maintenance of extracellular glutamate homeostasis. Recent human genetic studies have suggested that de novo mutations in GLT1 (EAAT2) cause early-onset epilepsy with multiple seizure types. Consistent with these findings, global GLT1 null mice show lethal spontaneous seizures. The consequences of GLT1 dysfunction vary between different brain regions, suggesting that the role of GLT1 dysfunction in epilepsy may also vary with brain regions. In this study, we generated region-specific GLT1 knockout mice by crossing floxed-GLT1 mice with mice that express the Cre recombinase in a particular domain of the ventricular zone. Selective deletion of GLT1 in the diencephalon, brainstem and spinal cord is sufficient to reproduce the phenotypes (excess mortality, decreased body weight, and lethal spontaneous seizure) of the global GLT1 null mice. By contrast, dorsal forebrain-specific GLT1 knockout mice showed nonlethal complex seizures including myoclonic jerks, hyperkinetic running, spasm and clonic convulsion via the activation of NMDA receptors during a limited period from P12 to P14 and selective neuronal death in cortical layer II/III and the hippocampus. Thus, GLT1 dysfunction in the dorsal forebrain is involved in the pathogenesis of infantile epilepsy and GLT1 in the diencephalon, brainstem and spinal cord may play a critical role in preventing seizure-induced sudden death.

Impaired striatal dopamine release in homozygous Vps35 D620N knock-in mice.

Point mutations in the vacuolar protein sorting 35 gene (VPS35) have been associated with an autosomal dominant form of late-onset Parkinson disease (PARK17), but there has been considerable debate over whether it is caused by a loss- or gain-of-function mechanism and over the intracellular target site of neurotoxicity. To investigate the pathogenesis of PARK17 in vivo, we generated Vps35 D620N knock-in (KI) mice, expressing the homologous mutant protein with endogenous patterns of expression, simultaneously with Vps35 deletion 1 (Del1) mice, which carry 1bp deletion in the exon15 of Vps35, by CRISPR/Cas9-mediated genome engineering. Neither homozygous nor heterozygous Vps35 D620N KI mice suffered from premature death or developed clear neurodegeneration up to 70 weeks of age. Vps35 Del1 allele appeared to be a null or at least severely hypomorphic allele and homozygous Vps35 Del1 showed early embryonic lethality. Heterozygous crossings between Del1 and D620N knock-in mice revealed that the D620N/Del1 compound heterozygous mice, but not heterozygous Del1 mice, suffered from survival disadvantage. In vivo microdialysis showed that DA release evoked by 120 mM potassium chloride was significantly reduced in the caudate putamen of adult homozygous Vps35 D620N KI mice. Taken together, these results suggest that Vps35 D620N allele is a partial-loss-of-function allele and that such a genetic predisposition and age-related alterations in the nigrostriatal dopamine system cooperatively influence the pathogenesis of PARK17.

Neuronal Heterotopias Affect the Activities of Distant Brain Areas and Lead to Behavioral Deficits.

Neuronal heterotopia refers to brain malformations resulting from deficits of neuronal migration. Individuals with heterotopias show a high incidence of neurological deficits, such as epilepsy. More recently, it has come to be recognized that focal heterotopias may also show a range of psychiatric problems, including cognitive and behavioral impairments. However, because focal heterotopias are not always located in the brain areas responsible for the symptoms, the causal relationship between the symptoms and heterotopias remains elusive. In this study, we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited spatial working memory deficit and low competitive dominance behavior, which have been shown to be closely associated with the activity of the medial prefrontal cortex (mPFC) in rodents. Analysis of the mPFC activity revealed that the immediate-early gene expression was decreased and the local field potentials of the mPFC were altered in the mice with heterotopias compared with the control mice. Moreover, activation of these ectopic and overlying sister neurons using the DREADD (designer receptor exclusively activated by designer drug) system improved the working memory deficits. These findings suggest that cortical regions containing focal heterotopias can affect distant brain regions and give rise to behavioral abnormalities. Significance statement: Recent studies reported that patients with heterotopias have a variety of clinical symptoms, such as cognitive disturbance, psychiatric symptoms, and autistic behavior. However, the causal relationship between the symptoms and heterotopias remains elusive. Here we showed that mice with focal heterotopias in the somatosensory cortex generated by in utero electroporation exhibited behavioral deficits that have been shown to be associated with the mPFC activity in rodents. The existence of heterotopias indeed altered the neural activities of the mPFC, and direct manipulation of the neural activity of the ectopic neurons and their sister neurons in the overlying cortex improved the behavioral deficit. Thus, our results indicate that focal heterotopias could affect the activities of distant brain areas and cause behavioral abnormalities.

Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ.

BACKGROUND: Although CRISPR/Cas enables one-step gene cassette knock-in, assembling targeting vectors containing long homology arms is a laborious process for high-throughput knock-in. We recently developed the CRISPR/Cas-based precise integration into the target chromosome (PITCh) system for a gene cassette knock-in without long homology arms mediated by microhomology-mediated end-joining. RESULTS: Here, we identified exonuclease 1 (Exo1) as an enhancer for PITCh in human cells. By combining the Exo1 and PITCh-directed donor vectors, we achieved convenient one-step knock-in of gene cassettes and floxed allele both in human cells and mouse zygotes. CONCLUSIONS: Our results provide a technical platform for high-throughput knock-in.

Glial dysfunction in the mouse habenula causes depressive-like behaviors and sleep disturbance.

The lateral habenula (LHb) regulates the activity of monoaminergic neurons in the brainstem. This area has recently attracted a surge of interest in psychiatry because studies have reported the pathological activation of the habenula in patients with major depression and in animal models. The LHb plays a significant role in the pathophysiology of depression; however, how habenular neurons are activated to cause various depression symptoms, such as reduced motivation and sleep disturbance, remain unclear. We hypothesized that dysfunctional astrocytes may cause LHb hyperactivity due to the defective uptake activity of extracellular glutamate, which induces depressive-like behaviors. We examined the activity of neurons in habenular pathways and performed behavioral and sleep analyses in mice with pharmacological and genetic inhibition of the activity of the glial glutamate transporter GLT-1 in the LHb. The habenula-specific inhibition of GLT-1 increased the neuronal firing rate and the level of c-Fos expression in the LHb. Mice with reduced GLT-1 activity in the habenula exhibited a depressive-like phenotype in the tail suspension and novelty-suppressed feeding tests. These animals also displayed increased susceptibility to chronic stress, displaying more frequent avoidant behavior without affecting locomotor activity in the open-field test. Intriguingly, the mice showed disinhibition of rapid eye movement sleep, which is a characteristic sleep pattern in patients with depression. These results provide evidence that disrupting glutamate clearance in habenular astrocytes increases neuronal excitability and depressive-like phenotypes in behaviors and sleep.

Reward-modulated motor information in identified striatum neurons.

It is widely accepted that dorsal striatum neurons participate in either the direct pathway (expressing dopamine D1 receptors) or the indirect pathway (expressing D2 receptors), controlling voluntary movements in an antagonistically balancing manner. The D1- and D2-expressing neurons are activated and inactivated, respectively, by dopamine released from substantia nigra neurons encoding reward expectation. However, little is known about the functional representation of motor information and its reward modulation in individual striatal neurons constituting the two pathways. In this study, we juxtacellularly recorded the spike activity of single neurons in the dorsolateral striatum of rats performing voluntary forelimb movement in a reward-predictable condition. Some of these neurons were identified morphologically by a combination of juxtacellular visualization and in situ hybridization for D1 mRNA. We found that the striatal neurons exhibited distinct functional activations before and during the forelimb movement, regardless of the expression of D1 mRNA. They were often positively, but rarely negatively, modulated by expecting a reward for the correct motor response. The positive reward modulation was independent of behavioral differences in motor performance. In contrast, regular-spiking and fast-spiking neurons in any layers of the motor cortex displayed only minor and unbiased reward modulation of their functional activation in relation to the execution of forelimb movement. Our results suggest that the direct and indirect pathway neurons cooperatively rather than antagonistically contribute to spatiotemporal control of voluntary movements, and that motor information is subcortically integrated with reward information through dopaminergic and other signals in the skeletomotor loop of the basal ganglia.

The synchronous activity of lateral habenular neurons is essential for regulating hippocampal theta oscillation.

Lateral habenula (LHb) has attracted growing interest as a regulator of serotonergic and dopaminergic neurons in the CNS. However, it remains unclear how the LHb modulates brain states in animals. To identify the neural substrates that are under the influence of LHb regulation, we examined the effects of rat LHb lesions on the hippocampal oscillatory activity associated with the transition of brain states. Our results showed that the LHb lesion shortened the theta activity duration both in anesthetized and sleeping rats. Furthermore, this inhibitory effect of LHb lesion on theta maintenance depended upon an intact serotonergic median raphe, suggesting that LHb activity plays an essential role in maintaining hippocampal theta oscillation via the serotonergic raphe. Multiunit recording of sleeping rats further revealed that firing of LHb neurons showed significant phase-locking activity at each theta oscillation cycle in the hippocampus. LHb neurons showing activity that was coordinated with that of the hippocampal theta were localized in the medial LHb division, which receives afferents from the diagonal band of Broca (DBB), a pacemaker region for the hippocampal theta oscillation. Thus, our findings indicate that the DBB may pace not only the hippocampus, but also the LHb, during rapid eye movement sleep. Since serotonin is known to negatively regulate theta oscillation in the hippocampus, phase-locking activity of the LHb neurons may act, under the influence of the DBB, to maintain the hippocampal theta oscillation by modulating the activity of serotonergic neurons.

Deep ultraviolet fluorescence microscopy of three-dimensional structures in the mouse brain.

Three-dimensional (3D) imaging at cellular resolution improves our understanding of the brain architecture and is crucial for structural and functional integration as well as for the understanding of normal and pathological conditions in the brain. We developed a wide-field fluorescent microscope for 3D imaging of the brain structures using deep ultraviolet (DUV) light. This microscope allowed fluorescence imaging with optical sectioning due to the large absorption at the surface of the tissue and hence low tissue penetration of DUV light. Multiple channels of fluorophore signals were detected using single or a combination of dyes emitting fluorescence in the visible range of spectrum upon DUV excitation. Combination of this DUV microscope with microcontroller-based motorized stage enabled wide-field imaging of a coronal section of the cerebral hemisphere in mouse for deciphering cytoarchitecture of each substructure in detail. We extended this by integrating vibrating microtome which allowed serial block-face imaging of the brain structure such as the habenula in mouse. Acquired images were with resolution high enough for quantification of the cell numbers and density in the mouse habenula. Upon block-face imaging of the tissues covering entire extent of the cerebral hemisphere of the mouse brain, acquired data were registered and segmented for quantification of cell number in each brain regions. Results in the current analysis indicated that this novel microscope could be a convenient tool for large-scale 3D analysis of the brain in mice.

GENETIC DELETION OF TRANSLOCATOR PROTEIN EXACERBATES POST-SEPSIS SYNDROME WITH ACTIVATION OF THE C1Q PATHWAY IN SEPTIC MOUSE MODEL.

ABSTRACT: Significant numbers of patients who survive sepsis exhibit psychiatric and cognitive impairments, termed post-sepsis syndrome. Understanding the underlying pathophysiology is essential to develop effective therapies. Translocator protein 18 kDa (TSPO) is a multifaceted mitochondrial protein implicated in inflammation, oxidative stress, and steroidogenesis in the central nervous system. Despite accumulated evidence demonstrating TSPO is a biomarker in psychiatric and neurodegenerative disorders, the role of this protein in post-sepsis syndrome remains elusive. The aim of this study was to investigate the role of TSPO in the long-term impairment of mouse behavior associated with psychiatric and cognitive impairments following sepsis induced by cecal ligation and puncture (CLP) surgery. Animals were divided into three groups: (i) wild type (WT) + sham, (ii) WT + CLP, and (iii) TSPO knock out + CLP. Survival rate and body weight change were assessed up to 17 days after surgeries. Then, we also assessed anxiety-like behavior, depression-like behavior, cognitive function, locomotor activity, and forelimb muscle strength in surviving mice by elevated plus maze, tail suspension test, y-maze, open field test, and grip strength test, respectively. Deletion of the TSPO gene led to high mortality and prolonged weight loss and exacerbated anxiety-like and depressive-like behavior with cognitive impairment 17 days after, but not before, CLP surgery. RNA-seq analysis of the hippocampus revealed the upregulation of genes (C1qb, C1qc, and Tyrobp) in C1q complement pathways correlated significantly with anxiety-like behavior that appeared long after CLP surgery. The expressions of these genes predicted other behavioral traits, including depressive-like behavior in the tail suspension test and grip power impairment, supporting the role of the C1q pathway in post-sepsis syndrome. Because the C1q pathway has recently attracted interest as a tag for pathological synaptic elimination, the current study suggests the C1q pathway is involved in the psychiatric and cognitive impairments observed in post-sepsis syndrome.

Neural mechanisms underlying uninstructed orofacial movements during reward-based learning behaviors.

During reward-based learning tasks, animals make orofacial movements that globally influence brain activity at the timings of reward expectation and acquisition. These orofacial movements are not explicitly instructed and typically appear along with goal-directed behaviors. Here, we show that reinforcing optogenetic stimulation of dopamine neurons in the ventral tegmental area (oDAS) in mice is sufficient to induce orofacial movements in the whiskers and nose without accompanying goal-directed behaviors. Pavlovian conditioning with a sensory cue and oDAS elicited cue-locked and oDAS-aligned orofacial movements, which were distinguishable by a machine-learning model. Inhibition or knockout of dopamine D1 receptors in the nucleus accumbens inhibited oDAS-induced motion but spared cue-locked motion, suggesting differential regulation of these two types of orofacial motions. In contrast, inactivation of the whisker primary motor cortex (wM1) abolished both types of orofacial movements. We found specific neuronal populations in wM1 representing either oDAS-aligned or cue-locked whisker movements. Notably, optogenetic stimulation of wM1 neurons successfully replicated these two types of movements. Our results thus suggest that accumbal D1-receptor-dependent and -independent neuronal signals converge in the wM1 for facilitating distinct uninstructed orofacial movements during a reward-based learning task.

Temporally regulated asymmetric neurogenesis causes left-right difference in the zebrafish habenular structures.

The habenular neurons on both sides of the zebrafish diencephalon show an asymmetric (laterotopic) axonal projection pattern into the interpeduncular nucleus. We previously revealed that the habenula could be subdivided into medial and lateral subnuclei, and a prominent left-right difference in the size ratio of these subnuclei accounts for the asymmetry in its neural connectivity. In the present study, birth date analysis showed that neural precursors for the lateral subnuclei were born at earlier stages than those for the medial subnuclei. More neurons for the early-born lateral subnuclei were generated on the left side, while more neurons for the late-born medial subnuclei were generated on the right side. Genetic hyperactivation and repression of Notch signaling revealed that differential timing determines both specificity and asymmetry in the neurogenesis of neural precursors for the habenular subnuclei.

Anti-seizure effects of medicinal plants in Malawi on pentylenetetrazole-induced seizures in zebrafish larvae.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants are used to manage and treat epilepsy in Malawi because of traditional beliefs and barriers to conventional anti-seizure drugs. Among the plants prescribed by traditional medical practitioners are Margaritaria discoidea, Dalbergia boehmii, Dalbergia nitidula, Catunaregam spinosa, and Lannea discolor. Despite the wide use of these plants, there is a lack of scientific evidence to support their anti-seizure efficacy. AIM OF THE STUDY: This study used the pentylenetetrazole (PTZ)-induced larval zebrafish seizure model to screen for anti-seizure effects of a collection of medicinal plants traditionally used in Malawi. MATERIALS AND METHODS: Zebrafish larvae were incubated in decoctions at maximum tolerated concentrations for 18 h and exposed to PTZ. As a primary screen, the effects of the decoctions on seizure-induced locomotor activity were determined. Decoctions that significantly reduced total distance traveled were further checked for effects on seizure latency and frequency, brain activity, immediate early gene expression, and c-fos protein expression. RESULTS: M. discoidea male leaves, D. boehmii roots, and D. nitidula leaves showed significant anti-seizure effects in the primary screen and were selected for further study. Electrophysiological and immediate early gene analyses corroborated anti-seizure effect of D. boehmii and D. nitidula. The results of c-fos protein expression further suggested that the anti-seizure effects in the larval brain may be mediated by the suppression of neurons localized in midbrain regions. CONCLUSIONS: These findings provide pioneering scientific evidence of the presence of anti-seizure activity in M. discoidea, D. boehmii, and D. nitidula, prescribed by traditional Malawian medical practitioners. Further studies are needed to identify and isolate compounds responsible for such biological activities and elucidate the possible mechanisms of action.

Identification of the zebrafish ventral habenula as a homolog of the mammalian lateral habenula.

The mammalian habenula consists of the medial and lateral habenulae. Recent behavioral and electrophysiological studies suggested that the lateral habenula plays a pivotal role in controlling motor and cognitive behaviors by influencing the activity of dopaminergic and serotonergic neurons. Despite the functional significance, manipulating neural activity in this pathway remains difficult because of the absence of a genetically accessible animal model such as zebrafish. To address the level of lateral habenula conservation in zebrafish, we applied the tract-tracing technique to GFP (green fluorescent protein)-expressing transgenic zebrafish to identify habenular neurons that project to the raphe nuclei, a major target of the mammalian lateral habenula. Axonal tracing in live and fixed fish showed projection of zebrafish ventral habenula axons to the ventral part of the median raphe, but not to the interpeduncular nucleus where the dorsal habenula projected. The ventral habenula expressed protocadherin 10a, a specific marker of the rat lateral habenula, whereas the dorsal habenula showed no such expression. Gene expression analyses revealed that the ventromedially positioned ventral habenula in the adult originated from the region of primordium lateral to the dorsal habenula during development. This suggested that zebrafish habenulae emerge during development with mediolateral orientation similar to that of the mammalian medial and lateral habenulae. These findings indicated that the lateral habenular pathways are evolutionarily conserved pathways and might control adaptive behaviors in vertebrates through the regulation of monoaminergic activities.

A Novel Microcontroller-Based System for the Wheel-Running Activity in Mice.

Voluntary wheel-running activity is a way to assess rodents' circadian rhythm and motivation for exercise. Deficits in these behaviors are implicated in the pathophysiology of sleep and psychiatric disorders. Limited space in animal facilities can hamper long-term monitoring of running wheel activity outside of the home cage. To address this issue, we provide a stand-alone solution to monitor the wheel-running activity of mice in their home cage. This system, named the wheel-running activity acquisition (WRAQ) system, is based on a microcontroller driven by a lithium polymer battery. With the WRAQ, we can record the wheel-running activity and illumination data for at least 30 d. Applying the WRAQ to an endotoxemia mouse model robustly detected the altered wheel-running activity and its recovery. With wireless data transfer capability extension, the system also allows for online monitoring and reporting of the circadian time (CT). We used the online monitoring of wheel-running activity with this extended WRAQ system and observed a significant shift of the active period in the circadian rhythm following a temporal chemogenetic activation of the suprachiasmatic nucleus (SCN)-subparaventricular zone (SPZ). Together, these findings indicate that the WRAQ system is a novel and cost-effective solution for the analysis of wheel-running activity in mice.

Activation of proprotein convertase in the mouse habenula causes depressive-like behaviors through remodeling of extracellular matrix.

The lateral habenula (LHb) attracts a growing interest as a regulator of monoaminergic activity which were frequently reported to be defective in depression. Here we found that chronic social defeat stress (CSDS) increased production of pro-inflammatory cytokines in LHb associated with mobilization of monocytes and remodeling of extracellular matrix by increased matrix metalloproteinase (MMP) activity. RNA-seq analysis identified proprotein convertase Pcsk5 as an upstream regulator of MMP activation, with upregulation in LHb neurons of mice with susceptibility to CSDS. PCSK5 facilitated motility of microglia in vitro by converting inactive pro-MMP14 and pro-MMP2 to their active forms, highlighting its role in mobilization of microglia and monocytes in neuroinflammation. Suppression of Pcsk5 expression via small interfering RNA (siRNA) ameliorated depressive-like behaviors and pathological mobilization of monocytes in mice with susceptibility to CSDS. PCSK5-MMPs signaling pathway could be a target for development of the antidepressants targeting the inflammatory response in specific brain regions implicated in depression.