Over a century ago Thorndike and Pavlov discovered that rewards need to follow the behaviors for conditioning. Recent studies indicate that dopamine release in the striatum serves as a reward signal and reinforces the preceding behaviors. However, the timing detection mechanisms of dopamine remains unknown. Using optical stimulation of glutamate and dopamine, we examined how dopamine affected the spike-timing plasticity (STDP) of single dendritic spines in the medium spiny neurons of the nucleus accumbens, a key brain area for reinforcement learning, and found that spine enlargement, representing long-term potentiation, was markedly enhanced only when dopamine was released within a very narrow time window (0.3-2 s) following the onset of STDP, consistent with the behavioral conditioning. We then performed imaging of Ca2+, CaMKII and PKA, and found that the sequence detection involved a process upstream of PKA activation: Sufficient generation of cAMP occurred only when spikes preceded dopamine to prime adenylyl-cyclase (AC1), otherwise cAMP was effectively removed by a potent phosphodiesterase (PDE) activity in thin distal dendrites of MSNs. Thus, PKA was activated only when spikes preceded dopamine, and promoted spine enlargement via CaMKII. Thus, we have clarified the cellular and molecular basis of reinforcement plasticity which is induced at the level of single dendritic spines.

Yagishita, S. et. al. A critical time window for dopamine actions on the structural plasticity of dendritic spines. Science 345:1616-1620, 2014. 

Yagishita, S. et. al. A critical time window for dopamine actions on the structural plasticity of dendritic spines. Science 345:1616-1620. 2014

Laboratory of Structural Physiology, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, The University of Tokyo

 Mammals exhibit a variety of physiological responses to psychological stress, such as increases in body temperature, heart rate and blood pressure.  These stress responses have a biological significance by increasing physical performances to survive a crisis of life, such as confrontation with enemies.  In the modern society, however, many people suffer from stress disorders caused by excessive psychological stressors.  For example, psychogenic fever caused by chronic psychological stress is a difficult psychosomatic symptom due to its resistance to antipyretics.

 We sought to determine the central circuit mechanism that drives stress-induced hyperthermia.  Rats given social defeat stress, a sociopsychological stress model, exhibited increases in sympathetic heat production by brown adipose tissue and in body temperature.  Drug nanoinjections into the brain revealed that activation of neurons in the dorsomedial hypothalamus (DMH) and rostral medullary raphe (rMR) mediates the stress-induced responses.  We also found that neurons projecting from the DMH to the rMR are activated by social defeat stress.  Specific stimulation of those projection neurons using an optogenetic technique elicited increases in brown adipose tissue thermogenesis, heart rate and blood pressure, mimicking stress responses.

 These results indicate that the direct pathway from the DMH to the rMR mediate the stress signaling that drives hyperthermia and other sympathetic responses (Figure).  We also found a stress-activated pathway from the DMH to the paraventricular hypothalamic nucleus, a neuroendocrine center for stress hormone release (Figure).  Our present findings may contribute to future development of therapies for stress disorders including psychogenic fever

Kataoka N, Hioki H, Kaneko T & Nakamura K (2014) Psychological stress activates a dorsomedial hypothalamus–medullary raphe circuit driving brown adipose tissue thermogenesis and hyperthermia. Cell Metabolism 20(2):346-358 (2014).

Figure: Schematic central circuits for sympathetic and neuroendocrine stress responses.  Forebrain stress signals activate two groups of DMH neurons: neurons in the dorsal part (dDMH) provide a direct glutamatergic input to sympathetic premotor neurons in the rMR to drive brown adipose tissue (BAT) thermogenesis contributing to hyperthermia, and neurons in the ventral part (vDMH) provide a direct input to the paraventricular hypothalamic nucleus (PVH) to drive a neuroendocrine outflow to release stress hormones.  Plus signs indicate excitatory neurotransmission.  IML, intermediolateral nucleus.

Career-Path Promotion Unit for Young Life Scientists, Kyoto University

In human heart, the slowly-activating delayed rectifier potassium current, also known as I_Ks, plays a key role in regulating cardiac excitability.  KCNQ1 and its auxiliary subunit KCNE1 underlie the I_Ks current by forming an ion channel complex.  Although KCNQ1 itself can produce potassium current, co-expression of KCNQ1 and KCNE1 produces slowly-activating and deactivating potassium current with a large positive shift (+40 mV) of the G-V curve.  In other words, KCNE1 makes KCNQ1 channel much harder to be activated, yet the molecular mechanism has been largely unknown.

            In this work, we found that Phe232 on the S4 segment (the center of voltage sensor) of KCNQ1 and Phe279 on the S5 segment from the neighbor KCNQ1 subunit are in proximity and collide during the upward movement of the voltage sensor during activation of the channel in the presence of KCNE1.  We also applied voltage clamp fluorometry to record the voltage sensor movement and the current simultaneously.  We found that the transition from the intermediate state (in which the voltage sensors are in the up state yet the pore is closed) to the open state is delayed due to the steric hindrance between the two phenylalanine residues (Phe232 and Phe279).

            These results indicate that the interaction between the voltage sensor domain and the pore domain of KCNQ1 is modified by the presence of KCNE1, and makes the channel harder to be activated.  Steric hindrance of the bulky phenylalanine residues plays a main role in creating the collision between the two domains.

Nakajo, K. & Kubo, Y. Steric hindrance between S4 and S5 of the KCNQ1/KCNE1 channel hampers pore opening. Nature Communications 5:4100, doi: 10.1038/ncomms5100 (2014). 

Figure. Two phenylalanine residues collide during activation of KCNQ1/KCNE1 channel

Figure. The phosphatase activity of voltage-sensing phosphatase (VSP) regulated by the movement of the voltage sensor. This study revealed that, at the single molecule level, the phosphatase activity of VSP is graded and the magnitude of the activity depends on the magnitude or mode of the movement of the voltage sensor. 

  1Integrative Physiology, Graduate School of Medicine, Osaka University, 2Institute for Academic Initiative, Osaka University

　P2X2 receptor channel, a homotrimer activated by binding of extracellular ATP to three intersubunit ATP binding sites (each located ~50Å from the ion permeation pore), also shows voltage-dependent activation upon hyperpolarization. Here, we used tandem trimeric constructs (TTC) harboring critical mutations at the ATP-binding, linker, and pore regions to investigate how ATP-activation signal is transmitted within the trimer and how signals generated by ATP and hyperpolarization converge. Analysis of voltage-and [ATP]-dependent gating in these TTCs showed that: (1) Voltage- and [ATP]-dependent gating of P2X2 requires binding of at least two ATP molecules. (2) D315A mutation in the beta-14 strand of the linker region connecting the ATP binding domains to the pore-forming helices induces two different gating modes; this requires presence of D315A mutation in at least two subunits. (3) T339S mutation in the pore domains of all three subunits abolishes the voltage dependence of P2X₂ gating in saturating [ATP], making P2X2 equally active at all membrane potentials. Increasing the number of T339S mutations in the TTC results in gradual changes in the voltage-dependence of gating from that of the wild-type channel, suggesting equal and independent contributions of the subunits at the pore level. (4) Voltage- and [ATP]-dependent gating in TTCs differs depending on the location of one D315A relative to one K308A that blocks the ATP binding and downstream signal transmission. (5) Voltage- and [ATP]-dependent gating does not depend on where one T339S is located relative to K308A (or D315A). Our results suggest that each intersubunit ATP binding signal is directly transmitted on the same subunit to the level of D315 via the domain that contributes K308 to the beta-14 strand. The signal subsequently spreads equally to all three subunits at the level of the pore, resulting in symmetric and independent contributions of the three subunits to pore opening.

Batu Keceli and Yoshihiro Kubo, Signal transmission within the P2X₂ trimeric receptor. Journal of General Physiology (2014) 143: 761-782 (Published online May 26 2014, 10.1085/jgp.201411166)  　

Figure. Schematic presentation of the activation-signal transmission from two ATP binding sites to the pore upon voltage- and [ATP]-dependent activation.

 For simplicity, two subunits are illustrated in red and blue. Colored spheres mark the K308 (orange) and K69 (blue) residues in the ATP binding region, D315 (yellow) in the linker, and T339 (green) at the pore level. Arrows with the same color of borderlines with the subunits depict the signal transmission on each subunit. The activation signal from one inter subunit ATP binding flows directly on the corresponding beta-14 strand of the ATP-binding site down to the D315 level, and then spreads to other subunits at the pore level.

Division of Biophysics and Neurobiology, Department of Molecular Physiology, National Institute for Physiological Sciences

The pH-dependent potassium channel, KcsA, has been extensively studied for the molecular mechanism of the gating. Crystallographic studies of the KcsA channel revealed the closed structure at neutral pH and the open structure at acidic pH. These crystallographic structures were obtained, however, for the channels extracted from the membrane (the detergent-solubilized form), thus, the native structure of the channel in the membrane-embedded condition still remains unsolved. In this study, we have directly observed the structure of the KcsA channel in the membrane using atomic force microscopy (AFM). With high-resolution imaging, we have successfully resolved the closed-gate structure at neutral pH and the open-gate structure at acidic pH. In the membrane, the open-gate channels were sparsely dispersed without contacting each other. On the other hand, we found surprisingly that the channels were clustered at neutral pH. To examine the time course of the clustering–dispersion dynamics we applied high-speed AFM. Upon pH changes, the channels underwent clustering–dispersion dynamics within several minutes, and reversible clustering–dispersion occurred for repeated pH changes. At acidic pH, a small fraction of the channels remained clustered, in which the channels did not open but changed the conformation slightly from the closed one (the intermediate gating structure). These results suggest that upon acidic change the channel undergoes the conformational change slightly in the cluster, and then opens the gate once the channel is dispersed as singly-isolated channels. This unprecedented collective behavior of the KcsA channel, gating-coupled clustering–dispersion in the membrane, gives clues of the complicated behavior of channel proteins on the cell membrane.

(Sumino, A., Yamamoto, D., Iwamoto, M., Dewa, T., Oiki, S.: Gating-Associated Clustering–Dispersion Dynamics of the KcsA Potassium Channel in a Lipid Membrane. J. Phys. Chem. Lett. 5: 578–584, 2014; Sumino, A., Sumikama, T., Iwamoto, M., Dewa, T., and Oiki, S.: The Open Gate Structure of the Membrane-Embedded KcsA Potassium Channel Viewed From the Cytoplasmic Side. Scientific Reports 3: 1063, 2013.)

Figure. Gating-coupled clustering–dispersion of the pH-dependent potassium channel, KcsA.

At neutral pH, the closed channels are clustered in which each channel is resolved as a round shape (left). Upon acidic change, the channels take slightly shortened but closed conformation in the cluster (the intermediate conformational state; center), and gradually they are dispersed as singly-isolated channels (right). The KcsA channel is a tetrameric channel, and the isolated channel has square shape with the pore at the center (an averaged image; right inset).

¹JST/PRESTO, ²Department of molecular physiology and biophysics, Faculty of medical sciences, University of Fukui

　Neuronal computation circuits are represented by brain structures. Ocular dominance columns (ODCs) have been well studied in the striate cortex (V1) of macaques, as well defined arrays of columnar structure. When ODCs were first identified in 1960’s, researchers thought that this structure represents circuit of stereoscopic depth coding. However, ODC expression seems obscure in some New World primate species, although they are all capable of stereopsis. Then, studies of depth coding through ODCs have been turned down. Previously, ODCs have been investigated by means of eye injections of transneuronal transporters and examination of cytochrome oxidase (CO) activity patterns after monocular enucleation. Here, we used the expression of immediate-early genes (IEGs), c-Fos and Zif268, after monocular inactivation (MI) to identify ODCs in New World owl monkeys. Using IEGs, we not only revealed apparent ODCs in owl monkeys but also discovered a number of unique features of their ODCs. These ODCs sometimes bridged to other columns in layer 4. The ODC pattern continued into V2. Finally, border strips were observed along ODC borders after only brief MI. Our data suggest that apparent species differences of ODCs were due to technical limitations, not fundamental nature, and revive debate over the functions, variability and development of ODCs.

 Takahata et al., Identification of ocular dominance domains in New World owl monkeys by immediate-early gene expression. Proc Natl Acad Sci USA, (2014) 111 (11): 4297-4302

Figure. A: In situ hybridization for c-Fos in a tangential section of flattened V1 of an owl monkey that was subjected for MI. Apparent ODCs were represented by the staining pattern.B: ODCs are not revealed by cytochrome oxidase (CO) staining that was commonly used to reveal ODCs. Adjacent sections were stained for c-Fos or CO. While c-Fos staining shows significant difference in relative optical density (ROD) between pale and dark columns, CO staining does not. Circles represent identical radial blood vessels of adjacent sections. 

 Department of Psychology, Vanderbilt University, USA

We are able to detect slight changes in the musical rhythm. Recent studies suggest that the cerebellum and basal ganglia are involved in temporal processing for non-motor cognitive functions (e.g. rhythm perception and temporal judgment; Fig. B). Here we show that neurons in the cerebellar dentate nucleus encode the interstimulus interval of isochronous rhythm and play a crucial role in predicting the timing of the next stimulus.

We trained monkeys to detect a single omission of isochronous repetitive stimuli (Fig. A). We found that the dentate neurons responded to each stimulus and gradually elevated the response as the repetition progressed, opposed to the sensory adaptation. The magnitude of the response positively correlated with the interstimulus interval (Fig. C). Because inactivation of the recording sites delayed the detection of stimulus omission, these signals might be necessary for the prediction of stimulus timing. This study revealed the underlying mechanisms of rhythm perception at single-neuron level. Our findings will advance the understanding of the cerebellar disorder and encourage future clinical techniques for the diagnosis and the evaluation of treatment.

Ohmae S, Uematsu A, Tanaka M. “Temporally specific sensory signals for the detection of stimulus omission in the primate deep cerebellar nuclei.” J Neurosci 2013; Sep 25

 (A) Missing oddball task. During monkeys looked at the fixation point (red dot) on the monitor, visual stimuli (white square) were presented repeatedly at a fixed interval. Tones were also presented simultaneously. As monkeys reported the omission of the audiovisual stimulus by making a saccadic eye movement, drops of juice were given as a reward. (B) Neural circuitry related to the temporal processing. Cb, Cerebellum; Thal, Thalamus; BG, Basal ganglia; Cortex, Cerebral cortex. (C) Example of a neuron recorded from the cerebellar dentate nucleus. In the left and right panels, data were aligned with the first stimulus and the omission of the stimulus, respectively. Vertical dashed line indicates the timing of each repetitive stimulus. Note that the response was greater as the repetition progressed and the interstimulus interval was longer. 

 1Department of Physiology, Hokkaido University School of Medicine (2Department of Psychology, University of Pennsylvania)

 Detecting threats and escaping before serious confrontations are important for animals to avoid danger and death. Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, is expressed in a subset of sensory neurons and mediates nociception evoked by pungent chemicals. Using behavioral testing, we found that TRPA1 knockout mice failed to avoid entering a chamber filled with vapor of formalin, allyl isothiocyanate, and acrolein. The avoidance behavior was blocked by nasal but not subcutaneous administration of a blocker to TRPA1. We also found that TRPA1 knockout mice did not wake when exposed to formalin during sleep. Additionally, the spinal trigeminal nucleus, the first relay neurons of the trigeminal system, showed massive expression of c-Fos after a brief (3 min) exposure to formalin vapor. TRPA1 seems to be a sentinel for environmental chemicals and induces avoidance behaviors and waking by way of the trigeminal system.

Yonemitsu T., et al., Sci Rep 3, 3100; DOI:10.1038/srep03100 (2013).

http://www.nature.com/srep/2013/131031/srep03100/full/srep03100.html

The basal ganglia control voluntary movements through three pathways: the direct, the indirect and the hyperdirect pathways (Figure A).  Our previous study showed that ablation of striatopallidal neurons in the indirect pathways (circled by red dashed lines in Figure A) by transgenic mice technique induced motor hyperactivity.  In the widely accepted model of the basal ganglia, the direct pathway is supposed to suppress the activity of the output station, the substantia nigra pars reticulata (SNr).  On the other hand, striatopallidal neurons are supposed to activate the SNr generally and suppress motor activity.

To elucidate the mechanism underlying the motor hyperactivity in striatopallidal ablated transgenic mice, we examined neuronal activity in the SNr.  The ablation of striatopallidal neurons has given little effect on spontaneous activity in the SNr.  However, response patterns in the SNr to motor cortical stimulation have been dramatically changed.  Motor cortical stimulation has induced a triphasic response composed of early excitation, inhibition and late excitation in normal condition (Figure B, left panel).  In contrast, the cortically evoked late excitation has been lost in striatopallidal ablated mice (Figure B, right panel).  Thus, the loss of the cortically evoked late excitation in the SNr seems to be responsible for motor hyperactivity.  These observations suggest that phasic late excitation in the SNr through striatopallidal neurons plays a key role to stop movements. 　Sano H., et al., J Neurosci, 33(17), 7583-7594, 2013

Figure legend

(A) The current model of basal ganglia circuitry.

The three pathways exert different effect on activity in the substantia nigra pars reticulata (SNr) and control of movements.

(B) Phasic late excitation in the SNr plays a key role to stop movements.

Motor cortical stimulation induces a triphasic response composed of early excitation, inhibition and late excitation in the SNr in the normal state.  The ablation of striatopallidal neurons diminishes late excitation in the SNr, and thus movements cannot be stopped, resulting motor hyperactivity.

The voltage-gated H⁺ channel functions as a dimer, a configuration that is different from standard tetrameric voltage-gated channels.  Each channel protomer has its own permeation pathway.  The C-terminal coiled-coil domain has been shown to be necessary both for dimerization and cooperative gating in the two channel protomers.  Here we report the gating cooperativity in trimeric and tetrameric Hv channels engineered by altering the hydrophobic core sequence of the coiled-coil assembly domain.  Trimeric and tetrameric channels exhibited more rapid and less sigmoidal kinetics of activation of H⁺ permeation than dimeric channels, suggesting that some channel protomers in trimer and tetramer failed to produce gating cooperativity observed in WT dimer.  Mulitimerization of trimer and tetramer channels were confirmed by protein-biochemical analyses including crystallography.  These findings indicate that the voltage-gated H⁺ channel is optimally designed as a dimeric channel, on a solid foundation of the sequence pattern of the coiled-coil core, with efficient cooperative-gating that ensures sustained and steep voltage-dependent H⁺ conductance in blood cells. 

Fujiwara Y, Kurokawa T, Takeshita K, Nakagawa A, Larsson HP, Okamura Y. “Gating of the Designed Trimeric/Tetrameric Voltage-Gated H⁺ Channel.” J Physiol 2013 591 (3) 627-640  Published on Cover. 

Ion channels form the ion-conducting pores on the lipid bilayer of the cell membrane and govern ion transport across the membrane by opening and closing the gate. This gate is operated not only by various types of stimuli from inside and outside of the cell, but also by membrane lipids around the ion channels. However, how the lipids affect the activity of ion channels remained unclear. To unveil the molecular mechanism of the lipid effect on the channel activity, we have analyzed the single-channel current of the KcsA potassium channel in the artificial membranes having various lipid compositions. We revealed that the KcsA channel was highly active when the anionic phospholipids exist in the inner leaflet. We identified that a protruding helix from the pore structure was the “membrane lipid sensor” of the KcsA channel. Furthermore, in the anionic lipid membrane, the rolled configuration of the lipid sensor was shown to stabilize the active form of the KcsA channel. (Iwamoto and Oiki, Proc. Natl. Acad. Sci. U. S. A. 110:749-754, 2013)

Figure; KcsA channels sense inner membrane lipids through the “membrane lipid sensor”. In the anionic lipid membrane, the rolled configuration of the sensor stabilizes the active form of the KcsA channel.

Department of Molecular Physiology and Biophysics, Faculty of Medical Sciences, University of Fukui

Yoshioka et al., Endothelial PI3K-C2a, a class II PI3K, has an essential role in angiogenesis and vascular barrier function.

Nature Medicine 2012 in press.

Department of Physiology, Kanazawa University School of Medicine

Calcium-dependent protein phosphatase calcineurin (CaN) is a key molecule to govern pathological cardiac hypertrophy. CaN dephosphorylates a downstream transcription factor NFAT, which in turn up-regulates hypertrophy-related genes. However, little is known about how CaN is activated independently of excitation-contraction coupling. Here, we found that the Na⁺/H⁺ exchanger NHE1 activates the CaN-NFAT signaling, leading to cardiomyocyte hypertrophy via direct binding of CaN to the 6-residues motif (PVITID) of NHE1. Over-expression of NHE1 promoted nuclear translocation and promoter activity of NFAT, which requires the clustering of NHE1 in lipid rafts. Importantly, increasing pH strongly activates CaN, thus we hypothesize that NHE1 produces a localized microdomain with higher pH, thereby sensitizing CaN to activation and promoting NFAT signaling. This is the first report showing unexpected functional coupling between pH-regulator NHE1 and Ca²⁺-dependent enzyme CaN.

Department of Molecular Physiology, National Cerebral and Cardiovascular Center

Parallel Mitral and Tufted Cell Pathways Route Distinct Odor Information to Different Targets in the Olfactory Cortex

The Journal of Neuroscience, June 6, 2012 • 32(23):7970 –7985

Department of Physiology, Graduate School of Medicine, The University of Tokyo

(*Present affiliation: Kavli Institute for Systems Neuroscience and Centre for the Biology of Memory, Norwegian University of Science and Technology)

Voltage-sensing phosphatase (VSP) consists of the two domains: voltage-sensor domain and the cytoplasmic region with phosphoinositide-phosphatase activities. The phosphatase region exhibits remarkable sequence similarity to PTEN, a tumor suppressor phosphatase. VSPs dephosphorylate the 5′ position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃] and phosphatidylinositol 4,5-bisphosphate [PI (4,5)P₂] upon voltage depolarization. However, it is unclear whether VSPs also have 3′ phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities with radiolabeled PI(3,4,5)P₃. TLC assay showed that the 3′ phosphate of PI(3,4,5)P₃ was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂] was removed by VSPs. Monitoring of PI(3,4)P₂ levels with the pleckstrin homology (PH) domain from tandem PH domain containing protein (TAPP1) fused with GFP (PH_TAPP1-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5′ phosphatase activity of VSP toward PI(3,4,5)P₃. However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P₃ is dephosphorylated at the 5′ position, PI(3,4)P₂ is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential.

Kurokawa T, Takasuga S, Sakata S, Yamaguchi S, Horie S, Homma KJ, Sasaki T, Okamura Y, PNAS, 109: 10089-10094 (2012)

 At presynaptic terminals, synaptic vesicle membranes are fused into plasma membrane upon exocytosis. After exocytosis, fused vesicle membranes are retrieved by endocytosis into presynaptic terminals to be reused. During sustained high frequency transmission, exo-endocytic coupling is critical for the maintenance of synaptic transmission. Here we show that this homeostatic coupling is supported by cGMP-dependent protein kinase (PKG) at the calyx of Held. Pharmacological tests with capacitance measurements revealed that presynaptic PKG activity accelerates vesicle endocytosis, and is supported by a retrograde signal cascade mediated by NO that is released by activation of postsynaptic NMDA receptors by the neurotransmitter glutamate. Activation of PKG also up-regulates phosphatidylinositol-4,5-bisphospate (PIP₂), thereby accelerating endocytosis. By accelerating vesicle endocytosis, presynaptic PKG activity supports synaptic fidelity during high frequency transmission. These mechanisms start to operate after hearing onset during the second postnatal week, when PKG expression becomes upregulated in the brainstem. We conclude that maturation of the PKG-dependent retrograde signal cascade strengthen the homeostatic plasticity for the maintenance of high frequency synaptic transmission at the calyx of Held synapse.

(Eguchi K, Nakanishi S, Takagi H, Taoufiq Z, Takahashi T. Neuron; 74: 517-29, 2012.)

 Figure: Retrograde regulation of vesicle endocytosis by nitric oxide