[show abstract][hide abstract] ABSTRACT: The brain demands high-energy supply and obstruction of blood flow causes rapid deterioration of the healthiness of brain cells. Two major events occur upon ischemia: acidosis and liberation of excess glutamate, which leads to excitotoxicity. However, cellular source of glutamate and its release mechanism upon ischemia remained unknown. Here we show a causal relationship between glial acidosis and neuronal excitotoxicity. As the major cation that flows through channelrhodopsin-2 (ChR2) is proton, this could be regarded as an optogenetic tool for instant intracellular acidification. Optical activation of ChR2 expressed in glial cells led to glial acidification and to release of glutamate. On the other hand, glial alkalization via optogenetic activation of a proton pump, archaerhodopsin (ArchT), led to cessation of glutamate release and to the relief of ischemic brain damage in vivo. Our results suggest that controlling glial pH may be an effective therapeutic strategy for intervention of ischemic brain damage.
[show abstract][hide abstract] ABSTRACT: Orexin/hypocretin neurons have a crucial role in the regulation of sleep and wakefulness. Recent optogenetic studies revealed that the activation or inhibition of orexin neuronal activity affects the probability of sleep/wakefulness transition in the acute phase. To expand our understanding of how orexin neurons maintain wakefulness, we generated new transgenic mice in which orexin neurons expressed archaerhodopsin from Halorubrum strain TP009 (ArchT), a green light-driven neuronal silencer, using the tet-off system (orexin-tTA; TetO ArchT mice). Slice patch clamp recordings of ArchT-expressing orexin neurons demonstrated that long-lasting photic illumination was able to silence the activity of orexin neurons. We further confirmed that green light illumination for 1 hr in the dark period suppressed orexin neuronal activity in vivo using c-Fos expression. Continuous 1 hr silencing of orexin neurons in freely moving orexin-tTA; TetO ArchT mice during the night (the active period, 20:00-21:00) significantly increased total time spent in slow-wave sleep (SWS) and decreased total wake time. Additionally, photic inhibition increased sleep/wakefulness state transitions, which is also evident in animals lacking the prepro-orexin gene, orexin neurons, or functional orexin-2 receptors. However, continuous 1 hr photic illumination produced little effect on sleep/wakefulness states during the day (the inactive period, 12:00-13:00). These results suggest that orexin neuronal activity plays a crucial role in the maintenance of wakefulness especially in the active phase in mice.
Behavioural brain research 05/2013; · 3.22 Impact Factor
[show abstract][hide abstract] ABSTRACT: Serotonergic (5HT) neurons of the dorsal raphe nuclei receive excitatory input from hypothalamic orexin (hypocretin) neurons and reciprocally inhibit orexin neurons through the 5HT1A receptor. However, the physiological significance of this negative feedback circuit for sleep/wakefulness regulation is little understood.
5HT1A receptor expression level was specifically and reversibly controlled in the orexin neurons using the Tet-off system. The responsiveness of orexin neurons to 5HT in vitro and the sleep/wakefulness patterns were compared between 5HT1A-overexpressing and control mice.
When the 5HT1A receptor was overexpressed in orexin neurons of Orexin-EGFP; orexin-tTA; TetO Htr1a mice, 5HT-induced inhibition of orexin neurons was prolonged. In the absence of doxycycline, Orexin-tTA; TetO Htr1a mice exhibited severe fragmentation of sleep/wakefulness during the first half of the dark period-the time of maximal activity in nocturnal rodents-without affecting sleep/wakefulness during the light period when sleep time is maximal. However, when the 5HT1A receptor in orexin neurons was reduced to basal expression levels in the presence of doxycycline, sleep/wakefulness patterns in Orexin-tTA; TetO Htr1a mice during the early active period were indistinguishable from those of littermate TetO Htr1a mice. These results strongly suggest that enhancement of inhibitory serotonergic input to orexin neurons caused fragmentation of wakefulness. In contrast, sleep/wakefulness architecture in the light period was unaffected by 5HT1A receptor overexpression in the orexin neurons.
Inhibitory serotonergic input likely functions as negative feedback to orexin neurons in the early dark period and helps stabilize wakefulness bouts, thereby contributing to the diurnal rhythm of sleep and wakefulness.
Tabuchi S; Tsunematsu T; Kilduff TS; Sugio S; Xu M; Tanaka KF; Takahashi S; Tominaga M; Yamanaka A. Influence of inhibitory serotonergic inputs to orexin/hypocretin neurons on the diurnal rhythm of sleep and wakefulness. SLEEP 2013;36(9):1391-1404.
[show abstract][hide abstract] ABSTRACT: Dynamic activity of glia has repeatedly been demonstrated, but if such activity is independent from neuronal activity, glia would not have any role in the information processing in the brain or in the generation of animal behavior. Evidence for neurons communicating with glia is solid, but the signaling pathway leading back from glial-to-neuronal activity was often difficult to study. Here, we introduced a transgenic mouse line in which channelrhodopsin-2, a light-gated cation channel, was expressed in astrocytes. Selective photostimulation of these astrocytes in vivo triggered neuronal activation. Using slice preparations, we show that glial photostimulation leads to release of glutamate, which was sufficient to activate AMPA receptors on Purkinje cells and to induce long-term depression of parallel fiber-to-Purkinje cell synapses through activation of metabotropic glutamate receptors. In contrast to neuronal synaptic vesicular release, glial activation likely causes preferential activation of extrasynaptic receptors that appose glial membrane. Finally, we show that neuronal activation by glial stimulation can lead to perturbation of cerebellar modulated motor behavior. These findings demonstrate that glia can modulate the tone of neuronal activity and behavior. This animal model is expected to be a potentially powerful approach to study the role of glia in brain function.
Proceedings of the National Academy of Sciences 11/2012; · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The genetic heterogeneity of autism poses a major challenge for identifying mechanism-based treatments. A number of rare mutations are associated with autism, and it is unclear whether these result in common neuronal alterations. Monogenic syndromes, such as fragile X, include autism as one of their multifaceted symptoms and have revealed specific defects in synaptic plasticity. We discovered an unexpected convergence of synaptic pathophysiology in a nonsyndromic form of autism with those in fragile X syndrome. Neuroligin-3 knockout mice (a model for nonsyndromic autism) exhibited disrupted heterosynaptic competition and perturbed metabotropic glutamate receptor-dependent synaptic plasticity, a hallmark of fragile X. These phenotypes could be rescued by reexpression of neuroligin-3 in juvenile mice, highlighting the possibility of reverting neuronal circuit alterations in autism after the completion of development.
[show abstract][hide abstract] ABSTRACT: Melanopsin (OPN4) is a photosensitive pigment originally found in a subtype of retinal ganglion cells and is a 7-transmembrane G-protein-coupled receptor (GPCR). Several previous reports showed that ectopic expression of OPN4 can be used as an optogenetic tool to control neural and cellular activities in various tissues. Compared with other optogenetic pigments, OPN4 is more sensitive to light, shows long-lasting activation, and can also control intracellular Ca(2+) dynamics. Here, we review how the ectopic expression of OPN4 enables the control of neural and cellular activities in vivo. In the retina, the ectopic expression of melanopsin in retinal ganglion cells successfully restored the vision of blind mice. It has also been reported that ectopic expression of melanopsin in orexin/hypocretin neurons enabled control of wakefulness in mice by blue light. In addition to neural activity, the ectopic expression of OPN4 has been reported to enable circuit control of the nuclear factor of activated T cells (NFAT) to enhance blood-glucose homeostasis in mice. We discuss the possibility of optogenetic control of other systems through the ectopic expression of OPN4.
Neuroscience Research 09/2012; · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Using in situ hybridization, we describe, for the first time, the profiles of expression of serotonin receptors (Htr/5-HTR) along the dorsal-ventral axis of mouse hippocampus. cRNA probes for most Htrs, excluding Htr6, were used. All hippocampal subregions and the entorhinal cortex cells providing input into the hippocampus were examined. The study shows that some, but not all, Htrs are expressed in the cells of the hippocampal circuitry. At both the subfield and the cell type levels, a somewhat overlapping pattern is observed. Four serotonin receptors, Htr1a, Htr2a, Htr2c and Htr7, display an expression pattern that changes along the dorsal-ventral axis of the hippocampus. Given the proposed functional differentiation of the hippocampus along its long axis, with the dorsal pole more involved in cognitive functions and the ventral pole more involved in mood and anxiety, our results suggest that serotonin receptors enriched in the ventral pole probably contribute to mood- and anxiety-related behaviours.
Philosophical Transactions of The Royal Society B Biological Sciences 09/2012; 367(1601):2395-401. · 6.23 Impact Factor
[show abstract][hide abstract] ABSTRACT: Melanopsin (OPN4) is a photosensitive G-protein-coupled photopigment and its ectopic expression enables control of neural activity induced by blue light. Here we report that we successfully expressed OPN4 in hypothalamic orexin/hypocretin neurons of double-transgenic mice (orexin-tTA; Bitet-O human OPN4 [hOPN4]/mCherry mice). In the double-transgenic mice, hypothalamic orexin neurons selectively expressed hOPN4 as well as mCherry as a reporter. We conducted slice patch-clamp recordings on hOPN4/mCherry-expressing orexin neurons, which showed long-lasting activation initiated by blue light even after the light was switched off. Optical fiber-guided blue light stimulation in the hypothalamus successfully initiated the electroencephalography pattern that reflects long-lasting wakefulness in the mice in vivo. Taken together, the results indicate that ectopic expression of hOPN4 in orexin neurons enables long-lasting activation of orexin neurons by blue light to control sleep/wakefulness of the mice.
Neuroscience Research 07/2012; · 2.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: Optogenetics has been enthusiastically pursued in recent neuroscience research, and the causal relationship between neural activity and behavior is becoming ever more accessible. Here, we established knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction (KENGE-tet) and succeeded in generating transgenic mice expressing a highly light-sensitive channelrhodopsin-2 mutant at levels sufficient to drive the activities of multiple cell types. This method requires two lines of mice: one that controls the pattern of expression and another that determines the protein to be produced. The generation of new lines of either type readily expands the repertoire to choose from. In addition to neurons, we were able to manipulate the activity of nonexcitable glial cells in vivo. This shows that our system is applicable not only to neuroscience but also to any biomedical study that requires understanding of how the activity of a selected population of cells propagates through the intricate organic systems.
[show abstract][hide abstract] ABSTRACT: Key points Autonomic respiratory rhythm is essential to maintain lives and is generated in the lower brainstem. The ventrolateral medullary region, called the pre-Bötzinger complex (preBötC), is the kernel for respiratory rhythm generation. Despite previous extensive studies focusing on neurons, the mechanism of how respiratory rhythm is generated has not been fully understood. Here we show that non-neuronal glial cells (a subset of putative astrocytes) in the preBötC are periodically activated preceding inspiratory neuronal activity, periodic activity of putative astrocytes persists during blockade of neuronal activity, and stimulation of astrocytes in the preBötC induces inspiratory neuronal firings. These findings together with the previous report that blockade of astrocytic metabolism abolishes inspiratory neural output suggest that astrocytes are functionally involved in respiratory rhythm generation. These results will help us better understand how respiratory rhythm is generated and how respiratory output is disturbed in various pathological conditions.
The Journal of Physiology 07/2012; 590(Pt 19):4933-44. · 4.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: Within the olfactory system, information flow from the periphery onto output mitral cells (MCs) of the olfactory bulb (OB) has been thought to be mediated by direct synaptic inputs from olfactory sensory neurons (OSNs). Here, we performed patch-clamp measurements in rat and mouse OB slices to investigate mechanisms of OSN signaling onto MCs, including the assumption of a direct path, using electrical and optogenetic stimulation methods that selectively activated OSNs. We found that MCs are in fact not typically activated by direct OSN inputs and instead require a multistep, diffuse mechanism involving another glutamatergic cell type, the tufted cells. The preference for a multistep mechanism reflects the fact that signals arising from direct OSN inputs are drastically shunted by connexin 36-mediated gap junctions on MCs, but not tufted cells. An OB circuit with tufted cells intermediate between OSNs and MCs suggests that considerable processing of olfactory information occurs before its reaching MCs.
Journal of Neuroscience 02/2012; 32(9):2964-75. · 6.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Optogenetics is a powerful neuromodulatory tool with many unique advantages to explore functions of neuronal circuits in physiology and diseases. Yet, interpretation of cellular and behavioral responses following in vivo optogenetic manipulation of brain activities in experimental animals often necessitates identification of photoactivated neurons with high spatial resolution. Although tracing expression of immediate early genes (IEGs) provides a convenient approach, neuronal activation is not always followed by specific induction of widely used neuronal activity markers like c-fos, Egr1 and Arc. In this study we performed unilateral optogenetic stimulation of the striatum in freely moving transgenic mice that expressed a channelrhodopsin-2 (ChR2) variant ChR2(C128S) in striatal medium spiny neurons (MSNs). We found that in vivo blue light stimulation significantly altered electrophysiological activity of striatal neurons and animal behaviors. To identify photoactivated neurons we then analyzed IEG expression patterns using in situ hybridization. Upon light illumination an induction of c-fos was not apparent whereas another neuronal IEG Npas4 was robustly induced in MSNs ipsilaterally. Our results demonstrate that tracing Npas4 mRNA expression following in vivo optogenetic modulation can be an effective tool for reliable and sensitive identification of activated MSNs in the mouse striatum.
PLoS ONE 01/2012; 7(12):e52783. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Mature oligodendrocytes are critical for myelin maintenance. To understand the molecular basis for this, genetic manipulation of mature oligodendrocytes is needed. Here we generated a mature oligodendrocyte tTA (tetracycline-controlled transcriptional activator) mouse line which, in combination with a tTA-dependent promoter line driving the expression of the desired transgene, can be used for gain-of-function studies. We used an oligodendrocyte promoter, the mouse proteolipid protein (PLP) promoter, to express mammalianized tTA, and generated a PLP-mtTA mouse line. In adults, mtTA mRNA was predominantly detected in brain white matter where it co-localized with PLP mRNA. mtTA-mediated gene induction was confirmed by crossing to mice with a tTA-dependent promoter driving expression of yellow fluorescent protein (tetO-YFP mice). YFP induction in PLP-mtTA::tetO-YFP mice was consistent with PLP expression in adult mature oligodendrocytes and premyelinating-stage myelinating oligodendrocytes. This PLP-mtTA mouse line is the first to enable gain-of-function studies in mature oligodendrocytes with the tet system.
[show abstract][hide abstract] ABSTRACT: Demyelination coincides with numerous changes of gene expression in the central nervous system (CNS). Cystatin F, which is a papain-like lysosomal cysteine proteinase inhibitor that is normally expressed by immune cells and not in the brain, is massively induced in the CNS during acute demyelination. We found that microglia, which are monocyte/macrophage-lineage cells in the CNS, express cystatin F only during demyelination. By using several demyelinating animal models and the spinal cord tissues from multiple sclerosis (MS) patients, we examined spatiotemporal expression pattern of cystatin F by in situ hybridization and immunohistochemistry. We found that the timing of cystatin F induction matches with ongoing demyelination, and the places with cystatin F expression overlapped with the remyelinating area. Most interestingly, cystatin F induction ceased in chronic demyelination, in which remyelinating ability was lost. These findings demonstrate that the expression of cystatin F indicates the occurrence of ongoing demyelination/remyelination and the absence of cystatin F expression indicates the cessation of remyelination in the demyelinating area.
Journal of Neuroscience Research 02/2011; 89(5):639-49. · 2.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: Signaling mediated by Notch receptors is crucial for the development of many organs and the maintenance of various stem cell populations. The activation of Notch signaling is first detectable by the expression of an effector gene, Hes5, in the neuroepithelium of mouse embryos at embryonic day (E) 8.0-8.5, and this activation is indispensable for the generation of neural stem cells. However, the molecular mechanism by which Hes5 expression is initiated in stem-producing cells remains unknown. We found that mammalian Gcm1 and Gcm2 (glial cells missing 1 and 2) are involved in the epigenetic regulation of Hes5 transcription by DNA demethylation independently of DNA replication. Loss of both Gcm genes and subsequent lack of Hes5 upregulation in the neuroepithelium of E7.5-8.5 Gcm1(-/-); Gcm2(-/-) mice resulted in the impaired induction of neural stem cells. Our data suggest that Hes5 expression is serially activated first by Gcms and later by the canonical Notch pathway.
[show abstract][hide abstract] ABSTRACT: Recent evidence indicates that leptin regulates appetite and energy expenditure, at least in part by inhibiting serotonin synthesis and release from brainstem neurons. To demonstrate that this pathway works postnatally, we used a conditional, brainstem-specific mouse CreER(T2) driver to show that leptin signals in brainstem neurons after birth to decrease appetite by inhibiting serotonin synthesis. Cell-specific gene deletion experiments and intracerebroventricular leptin infusions reveal that serotonin signals in arcuate nuclei of the hypothalamus through the Htr1a receptor to favor food intake and that this serotonin function requires the expression of Creb, which regulates the expression of several genes affecting appetite. Accordingly, a specific antagonist of the Htr1a receptor decreases food intake in leptin-deficient but not in Htr1a(-/-) mice. Collectively, these results establish that leptin inhibition of serotonin is necessary to inhibit appetite postnatally and provide a proof of principle that selective inhibition of this pathway may be a viable option to treat appetite disorders.
Journal of Experimental Medicine 01/2011; 208(1):41-52. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dopamine system disorders ranging from movement disorders to addiction and schizophrenia involve striatal medium spiny neurons (MSNs), yet their functional connectivity has been difficult to determine comprehensively. We generated a mouse with conditional channelrhodopsin-2 expression restricted to medium spiny neurons and assessed the specificity and strength of their intrinsic connections in the striatum and their projections to the globus pallidus and the substantia nigra. In the striatum, medium spiny neurons connected with other MSNs and tonically active cholinergic interneurons, but not with fast-spiking GABA interneurons. In the globus pallidus, medium spiny neurons connected strongly with one class of electrophysiologically identified neurons, but weakly with the other. In the substantia nigra, medium spiny neurons connected strongly with GABA, but not with dopamine neurons. Projections to the globus pallidus showed solely D2-mediated presynaptic inhibition, whereas projections to the substantia nigra showed solely D1-mediated presynaptic facilitation. This optogenetic approach defines the functional connectome of the striatal medium spiny neuron.
Journal of Neuroscience 01/2011; 31(4):1183-92. · 6.91 Impact Factor