Published papers

Serotonin is a neuromodulator that is involved extensively in behavioral, affective, and cognitive functions in the brain. Previous recording studies of the midbrain dorsal raphe nucleus (DRN) revealed that the activation of putative serotonin neurons correlates with the levels of behavioral arousal [1], rhythmic motor outputs [2], salient sensory stimuli [3-6], reward, and conditioned cues [5-8]. The classic theory on serotonin states that it opposes dopamine and inhibits behaviors when aversive events are predicted [9-14]. However, the therapeutic effects of serotonin signal-enhancing medications have been difficult to reconcile with this theory [15, 16]. In contrast, a more recent theory states that serotonin facilitates long-term optimal behaviors and suppresses impulsive behaviors [17-21]. To test these theories, we developed optogenetic mice that selectively express channelrhodopsin in serotonin neurons and tested how the activation of serotonergic neurons in the DRN affects animal behavior during a delayed reward task. The activation of serotonin neurons reduced the premature cessation of waiting for conditioned cues and food rewards. In reward omission trials, serotonin neuron stimulation prolonged the time animals spent waiting. This effect was observed specifically when the animal was engaged in deciding whether to keep waiting and was not due to motor inhibition. Control experiments showed that the prolonged waiting times observed with optogenetic stimulation were not due to behavioral inhibition or the reinforcing effects of serotonergic activation. These results show, for the first time, that the timed activation of serotonin neurons during waiting promotes animals patience to wait for a delayed reward.

Astrocytes generate local calcium (Ca2+) signals that are thought to regulate their functions. Visualization of these signals in the intact brain requires an imaging method with high spatiotemporal resolution. Here, we describe such a method using transgenic mice expressing the ultrasensitive ratiometric Ca2+ indicator yellow Cameleon-Nano 50 (YC-Nano50) inastrocytes. In these mice, we detected a unique pattern of Ca2+ signals. These occur spontaneously, predominantly in astrocytic fine processes, but not the cell body. Upon sensory stimulation, astrocytes initially responded with Ca2+ signals at fine processes, which then propagated to the cell body. These observations suggest that astrocytic fine processes function as a high-sensitivity detector of neuronal activities. Thus, the method provides a useful tool for studying the activity of astrocytes in brain physiology and pathology. © 2014 The Authors.

Memory traces are believed to be ensembles of cells used to store memories. To visualize memory traces, we created a transgenic line that allows for the comparison between cells activated during encoding and expression of a memory. Mice re-exposed to a fear-inducing context froze more and had a greater percentage of reactivated cells in the dentate gyrus (DG) and CA3 than mice exposed to a novel context. Over time, these differences disappeared, in keeping with the observation that memories become generalized. Optogenetically silencing DG or CA3 cells that were recruited during encoding of a fear-inducing context prevented expression of the corresponding memory. Mice with reduced neurogenesis displayed less contextual memory and less reactivation in CA3but, surprisingly, normal reactivation in the DG. These studies suggest that distinct memory traces are located in the DG and in CA3 but that the strength of the memory is related to reactivation in CA3. © 2014 Elsevier Inc.

Whether increased serotonin (5-HT) release in the forebrain attenuates or enhances anxiety has been controversial for over 25 yr. Although there is considerable indirect evidence, there is no direct evidence that indicates a relationship between acute 5-HT release and anxiety. In particular, there is no known method that can reversibly, selectively, and temporally control serotonergic activity. To address this issue, we generated transgenic animals to manipulate the firing rates of central 5-HT neurons by optogenetic methods. Activation of serotonergic neurons in the median raphe nucleus was correlated to enhanced anxiety-like behaviour in mice, whereas activation of serotonergic neurons in the dorsal raphe nucleus had no effect on anxiety-like behaviour. These results indicate that an acute increase in 5-HT release from the median raphe nucleus enhances anxiety.

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. © 2014 Elsevier Inc.

Optogenetics, the use of channelrhodopsin: genetically-encodable light-activated proteins, allows the manipulation of specific neural circuit elements with millisecond precision. It has been 10 years since the discovery of channelrhodopsin, and optogenetics is now widely accepted as a powerful tool for addressing causal relationships between neural activity and behavior.

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. h in the dark period suppressed orexin neuronal activity in vivo using c-Fos expression. Continuous 1. h 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. h 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. © 2013 The Authors.

Study Objective: 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. Design: 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. Measurements and Results: When the 5HT1A receptor was overexpressed in orexin neurons of Orexin-EGFP; orexin-tTA; TetO Htr1a mice, 5HTinduced 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. Conclusion: 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. © 2013 Associated Professional Sleep Societies, LLC.

Astrocytes, a major subtype of glia, interact with neurons as a supportive partner supplying energy sources and growth factors. Astrocytes regulate the activity of neighboring neurons by releasing chemical transmitters (gliotransmitters). However, the precise role of gilotransmitters in regulating neuronal activity is still under debate. Here, we report that a subtle enhancement in the release of one gliotransmitter, ATP, affects synaptic potentiation from an analysis of mice containing an astrocyte-selective (GFAP) mutation. We found that, relative to normal mice, weaker stimulation induced long-term potentiation (LTP) in mutant mice, indicating that the threshold to induce LTP was lowered in the mutant. While excitatory transmission was normal in the mutant, inhibitory GABAergic transmission was suppressed. We found that a low concentration of adenosine selectively attenuated inhibitory neuronal activity and lowered the threshold to induce LTP in wild type mice. In comparison, adenosine A1 receptor antagonism reversed the lowered LTP threshold back to normal in the mutant mouse. We verified that adenosine levels in the cerebrospinal fluid of mutant mice were slightly elevated compared to wild type mice. This was apparently caused by an increase in ATP release from mutant astrocytes that could provide a source of augmented adenosine levels in the mutant. ATP is thought to suppress the excitability of neuronal circuits; however, a small increase in ATP release can result in a suppressed inhibitory tone and enhanced excitability of neuronal circuitry. These findings demonstrate that ATP released from astrocytes acts in a bidirectional fashion to regulate neuronal excitability depending on concentration. © 2012 Wiley Periodicals, Inc.