生理学研究所年報

8. 中枢神経系における局所回路とその機能特性

2000年11月21日−11月22日
代表・世話人：三上　章允（京都大学霊長類研究所)
所内対応者：森　茂美（生理学研究所)

（１）: Local circuit in the cerebral cortex
Takeshi Kaneko(Kyoto Univ., Japan)
（２）: Fast neuronal population oscillations in cortical networks in vitro
Eberhard H. Buhl(Univ. of Leeds, UK)
（３）: Functional subdivisions of cortical neurons: Do interstitial cells in the white matter participate
in the cortical circuit?
Nobuaki Tamamaki(Kyoto Univ., Japan)
（４）: Anatomy, physiology, molecular and circuit properties of neocortical interneurons
Henry Markram, Wang Yun, Anirudh Gupta & Maria Toledo (The Weizmannn Institute of Science, Israel)
（５）: Depolarizing afterpotentials involved in rhythmogenesis in cortical pyramidal cells
Youngnam Kang and Yo Kishi (Kyoto Univ., & Health Sci. Univ. of Hokkaido , Japan)
（６）: Electrophysiology and morphology of rat claustral neurons
Hiroshi Shibuyaand Tetsuro Yamamoto (Mie Univ., Japan)
（７）: Synaptic connections and synchronized firing of cortical interneurons
Yasuo Kawaguchi, Yoshiyuki Kubota and Satoru Kondo(National Inst. for Physiol. Sci., & RIKEN, Japan)
（８）: Electrical synapses and networks of GABAergic neurons in the neocortex
Shaul Hestrin and Mario Galarreta (Stanford Univ., USA)
（９）: Local circuit and representation of sound
Wen-Jie Song and Hideo Kawaguchi (Osaka Univ., & Hitachi, Ltd., Japan)
（10）: Classification of neuron types based on the firing patterns recorded in behaving monkeys
Akichika Mikami, Keichiro Kato, Shunpei Unno and Yungnum Kang(Kyoto Univ., Japan)
（11）: Velocity and position guided orienting in unrestrained cats
Shigeto Sasaki, Kazuya Yoshimura and Kimisato Natito (Tokyo Metropolitan Inst. for Neurosci., Japan)
（12）: Dynamic regulation of signal transmission in the local circuit of the superior colliculus
Yasuhiko Saito and Tadashi Isa (National Inst. for Physiol. Sci., Japan)
（13）: Mutual inhibition in the premotor circuit controlling saccade
Kaoru Yoshida, Y. Iwamoto, Sohei Chimoto and Hiroshi Shimazu (Univ. of Tsukuba, Japan)
（14）: Synchronous oscillatory activity of local field potentials and neurons in MI and SMA
during bimanual movements of monkeys
Takashi Kawashima, Thomas A. Knight and Eberhard E. Fetz (Gifu Univ., & Unv. of Washington, USA)
（15）: A new bipedally walking monkey model for the study of human bipedal locomotion
Shigemi Mori, Futoshi Mori, Katsumi Nakajima, Atsumichi Tachibana, Masahiro Mori and Chijiko Takasu 　(National Inst. for Physiol. Sci., Japan)
（16）: Selective expression of Ca²⁺－permeable AMPA receptors in horizontal cells in the superficial layer of rat superior colliculus
Endo, T. and Isa, T. (National Inst. For Physiol. Sci., Japan)
（17）: Burst firing in layer III pyramidal neurons of cat sensorimotor cortex
Nishimura, Y., Shibuya, H., Asahara, T. and Yamamoto, T. (Mie Univ., Japan)
（18）: Interaction of horizontal and vertical inputs to pyramidal cells in the superficial layers of the cat visual cortex
Sato, H., Yoshimura, Y., Imamura, K. and Watanabe, Y. (Osaka Univ., Osaka Biosci. Inst. & Nagoya Univ., Japan)
（19）: Spatiotemporal interaction of response to multiwhisker stimulation in the barrel cortex neurons of rats
Shimegi, S., Akasaki, T. and Sato, H. (Osaka Univ., Japan)
（20）: Functional Development of the Recurrent Inhibitory Network of the Mauthner Cell in Zebrafish larvae
Takahashi, M., Narushima, M., Murayama, Y. and Oda, Y. (Osaka Univ., Japan)
（21）: Fluorescence imaging of the protein kinase C translocation in the hippocampal neurons
Tsubokawa, H. and Takagi, S. (National Inst. For Physiol. Sci., Japan)
（22）: Calciumu-sensitive cation current accompanying activation of nicotinic acetylcholine receptors
in rat mesopontine dopamine neurons
Yamashita, T. and Isa, T. (National Inst. For Physiol. Sci., Japan)

【参加者名】
　E. Buhl (University of Leeds)，H. Markram (University of California San Francisco)，S. Hestrin (Stanford Universi- ty)，森　茂美（生理学研究所），金子　武嗣（京都大学大学院・医学研究科・高次脳科学講座），玉巻　伸章（京都大学大学院・医学研究科・高次脳科学講座），姜　英男（北海道医療大学），山本　哲朗（三重大学・医学部・生理学第二講座），西村　嘉洋（三重大学・医学部・生理学第二講座），渋谷　浩司（三重大学・医学部・生理学第二講座），吉田　薫（筑波大学・基礎医学系・生理），川口　泰雄（生理学研究所・大脳神経回路論研究部門），根東　覚（生理学研究所・大脳神経回路論研究部門），窪田芳之（理化学研究所），苅部冬紀（理化学研究所），宋　文杰（大阪大学・基礎工学部），川口英夫（日立基礎研究所），Oo HaiLiang（大阪大学・基礎工学部），佐々木成人（財）東京都神経科学総合研究所・神経生理学部門），内藤　公郷（財）東京都神経科学総合研究所・システム生理），伊佐　正（生理学研究所・統合生理研究施設高次脳機能研究プロジェクト），斎藤　康彦（生理学研究所・統合生理研究施設高次脳機能研究プロジェクト），遠藤　利朗（生理学研究所・統合生理研究施設高次脳機能研究プロジェクト），山下　哲司（生理学研究所・統合生理研究施設高次脳機能研究プロジェクト），坪川　宏（生理学研究所・脳機能計測センター），高木佐知子（生理学研究所・脳機能計測センター），深井　朋樹（玉川大学・工学部），川島卓（岐阜大学・医学部・反射研究施設），三上　章允（京都大学・霊長類研究所・行動神経研究部門），海野　俊平（京都大学・霊長類研究所・行動神経研究部門），猿渡　正則（京都大学・霊長類研究所・行動神経研究部門），加藤啓一郎（京都大学・霊長類研究所・行動神経研究部門），山口　峻司（山形大学・理工学研究科），伊豆田　義人（山形大学・理工学研究科），増田　健（山形大学・理工学研究科），浜田　生馬（財）東京都神経科学総合研究所・神経生理学部門），中村　泰尚（東京医科歯科大学大学院・医歯学総合研究科・神経解剖学分野），村越　隆之（東京医科歯科大学・医学部・薬理），田中　昌司（上智大学），佐藤宏道（大阪大学・健康体育部），七五三木聡（大阪大学・健康体育部），赤碕孝文（大阪大学・健康体育部），尾関宏文（大阪大学・健康体育部），田谷文彦（大阪大学・健康体育部），定金　理（大阪大学・健康体育部），木田裕之（大阪大学・健康体育部），村越　隆之（東京医科歯科大学・医学部・薬理学），高橋　正治（大阪大学・基礎工学部），駒井　章治（神戸大学・生理学第一講座），武田　湖太郎（慶應義塾大学・大学院理工学研究科・基礎理工学専攻），林　民（三重大学・医学部・生理学第二講座），中世古幸成（三重大学・医学部・生理学第二講座），一戸（弘前大学），木村（大阪大学），青柳（京都大学），伊藤（京都産業大学），藤井（京都産業大学），伊藤（愛知学院大学）

【概要】
　中枢神経系では，異なる機能特性を持つ比較的少数の神経細胞の構成する規則的で局所的な回路が重要な役割を演じている。局所的な神経回路は機能単位を構成し，この機能単位相互の情報交換と，遠方との神経結合による情報交換が中枢神経系における複雑な情報処理の基礎となっている。このような過程の解明は，一人の研究者，一つの研究室で取り組むには膨大すぎる。本研究会では，分子から個体に至る異なる研究レベル，異なる手法による各研究室の取り組みを持ちより，中枢神経系における局所回路とその機能特性について討議し，こうした視点からの研究の推進を目指した。平成10年度から3年間の計画で取り組んだ研究会は，平成12年度，その最終年度に当たる。そこで今回は，文部省科学研究費特定領域研究A「脳研究の総合的推進に関する研究」の支援を受け，3人の海外からの参加者を招聘し，発表と討論はすべて英語で行った。海外からの参加者が現在活躍中の第一級の研究者であったことも幸いし，多くの参加者を得て活発な討論が行われた。特にHenry Markramの膨大なデータと細胞タイプの詳細な分類には圧倒された。また，口頭発表のほかポスター発表も行い，若い研究者を中心に海外からの参加者や他の研究室の研究者との交流の場となった。本研究会が当初目指していた大型の研究プロジェクトの実現は起動に乗らなかったが，研究会の中から共同研究なども育ち，神経回路に依拠した機能研究の推進に一定の役割を果たすことができた。

(1) Local circuitin the cerebral cortex

T. Kaneko (Department of Physiology, Faculty of Medicine, Kyoto University)

    To reveal the mechanism of information processing in the cerebral cortex, we focused on the morphological analysis of its intrinsic circuitry. Recently, we developed a retrograde neuronal tracing method with Golgi-like soma- dendritic filling. By combining this method with the intra- cellular staining technique, local connection from layer III pyramidal cells to corticospinal and corticothalamic projection neurons were examined in the motor cortical slices of the rat.
    Forty layer III pyramidal neurons stained intracellularly were of the regular spiking type, showed immunoreactivity for glutaminase, and emitted axon collaterals arborizing local-ly in layers II/III and /or V. Nine of them were reconstruct- ed for morphological analysis; 15.2% or 3.8% of varicosities of axon collaterals of the reconstructed neurons were apposed to dendrites of corticospinal or corticothalamic neurons,respectively. By con-focal laserscanning and electron micro- scopy, some of these appositions were revealed to make synapses. These findngs indicate that corticospinal neurons receive information from the superficial cortical layers 4-fold more efficiently than corticothalamic neurons.
    The connections were further examined by intracellular recording of excitatory postsynaptic potential (EPSP) that were evoked in layer V and layer VI pyramidal neurons by stimulation of layer II/III. EPSPs evoked in layer V pyramidal neurons showed short and constant onset latencies, suggest- ing their monosynaptic nature. In contrast, most EPSPs evokedin layer VI pyramidal neurons had long onset latencies and showed double-shock facilitation of onset latency, implying that they were polysynaptic.
    The results suggest that information of the superficial cortical layers is transferred directly and efficiently to corticospinal neurons in layer V and thereby exerts an im- portant influence on cortical motor output. Corticothalmic neurons are, in contrast, considered relatively independent of or indirectly related to information processing of the superficial cortical layers.

(2) Fast neuronal population oscillations in cortical networks in vitro

Eberhard H. Buhl (The School of Biomedical Sciences, University of Leeds, United Kingdom)

Many neurons in the CNS are endowed with oscilla- tory properties which allow them to discharge rhythmically and, thus, entrain their neighbours and distant projection targets alike. However, rhythmic population oscillations may also arise as an emergent property of a neuronal network which is suitably interconnected with electrical and/or chemical synapses. It is feasible to analyse thetiming of action potentials and synaptic events in pyramidal cells and interneurons by using the concomitantly recorded extracellular field as a temporal reference. It thus emerges that the discharge probability of pyramidal neurons in- creases concomitantly with the decay of inhibitory currents /potentials.While there is little doubt with respect to the role of synaptic mechanisms in governing the generation of synchronous activity, equally compelling evidence has been accruing that, in the absence of synaptic communication, gap junction-mediated electrical signalling is effective in gen- erating network oscillations. Spontaneous, ultrafast (~200 Hz) ripples can, for example, be observed in hippocampal tissue in vitro. Moreover, transient depolarizations with high-molarity potassium solutions can trigger fast (~100 Hz) synchronous network activity in either nominally calcium- free solutions or the presence of GABA and glutamate receptor antagonists. Interestingly, when leaving synaptic transmission intact, such fast rhythms appear to be nested (i.e. phase-locked) in a slower gamma frequency population oscillation. These data therefore support the notion of non- synaptic (presumably a mixture of electrical and ephaptic effects) and synaptic mechanisms showing a complex interplay in the generation of complex neuronal rhythms which frequently, both in vivo and in vitro, span more than a single EEG frequency band.

(3) Functional subdivisions of cortical neurons:
"Do interstitial cells in the white matter participate in the cortical circuit?"

Tamamaki, N. (Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University)

Interstitial cells in the white matter of the neocortex is a remnant population of early generated neurons found in the intermediate zone of the developing neocortex of the rat. Most of these early generated neurons were reported as GABAergic neurons and might work as pioneer neurons projecting subcortically. Most of those early generatedneurons were eliminated by cell death after birth. However, some of them remained in the white matter and retained axons and dendrites. We revealed the axon and dendritic arbors of these interstitial cells by recombinant adenovirus infection followed by modified-GFP exrpession. We could divide the interstitial cells into two types and one of them were Martinotti cells. The other type was not described so far and regarded a new type of inter neurons of the neocortex. The new type of the interstitial cells had an axon arborized preferentially in the white matter. The terminals may be targeting on edges of basal dendrites or axons in the white matter.

(4) Anatomy, Physiology, Molecular and Circuit Properties of Neocortical Interneurons

Henry Markram, Wang Yun, Anirudh Gupta & Maria Toledo
(Department of Neurobiology, The Weizmannn Institute of Science, Israel)

Interneurons comprise a small fraction of the total neurons of the neocortex, but are critical for normal function. In this study we examined in detail a large number of interneurons in the somatosensory cortex of rats and developed methods to allow objective classification of different types of interneurons in terms of their anatomy and physiology. Five major anatomical classes were examined: Large Basket Cells, Nest Basket Cells, Small Basket Cells, Martinotti Cells and Bitufted Cells. We derived a comprehensive morphology code (mCode) that was used to examine the similarities and differences between LBCs, NBCs and SBCs. Clear anatomical identification of interneurons allowed examination of electrophysiological subtypes and we found that most interneurons expressed 1-5 subtypes of electrophysiological behaviors. We further examined the molecular expression patterns of these anato- physiological subtypes, and found that most subtypes expressed in 2-5 different ways. We also ventured to estimate the extent of the diversity of interneurons. In total, we predict that at least 15 anatomical classes of interneurons exist in each of layers 2-6, with an average of 3 electrophysiological subtypes each and of these a further average of 4molecular subtypes each. This suggests that each neocortical layer could contain around 180 different types of interneurons. The distribution of different types of interneurons varies from 1:1 to 1:20 with an average of around 1:5, indicating that around 675 interneurons are required in a given layer to provide comprehensive diversity of interneurons. The ratio of interneuron numbers across layers also varies on average 2 fold, suggesting that a column of cortex would need to hold around 9,000 interneurons or around 76,000 neurons. This is equivalent to about 7 cortical columns of diameter 300 mm² or 0.63 mm² of cortical surface.

(5) Depolarizing afterpotentials involved in rhythmogenesis in cortical pyramidal cells

Youngnam Kang (Department of Physiology, Faculty of Medicine, Kyoto University,
Department of Oral Physiology, Faculty of Dentistry, Hokkaido Health Science University,)
Yo Kishi (Department of Physiology, Faculty of Medicine, Kyoto University)

Regular spiking cortical pyramidal cells often display an early depolarizing spike afterpotentials (early DAP) immediately following spike repolarization, and an enhance- ment of the DAP led to a generation of fast rhythmic burst firing (FRB). This FRB is generally thought to underlie gamma-band EEG oscillation. On the other hand, the late DAP is also seen following spike-AHP in regular spiking pyramidal cells when metabotropic glutamate or muscarinic receptors are activated. This late DAP has been suggested to be associated with theta EEG rhythm. We have investi- gated the ionic mechanisms underlying these early and late DAPs. The data suggest that the enhancement of the early DAP is mediated by an enhancement of the sensitivity of cat- ionic channels to Ca²⁺ through the activation of CaMKII. On the other hand, we conclude that in the presence of muscarine, calcium influx through voltage-gated calcium channels triggers IP3 induced calcium release leading to a generation of calcium- dependent late DAP and excessive clacium influx results in a suppression of the late DAP through inactivation of IP3- receptors by the calcium-calmodulin complexes.

(6) Electrophysioology and morphology of rat claustral neurons

Hiroshi Shibuya and Tetsuro Yamamoto (Department of Physiology, Faculty of Medicine, Mie University)

The electrophysiological and morphological properties of constituent neurons and fiber connections of the rat rostral claustrum gave some suggestions about its ontoge- netic origin and function. All recorded neurons had action potentials with much higher maximum rates of rise than that of fall, and spike-frequency adaptation during long depolarizing pulses that could be classified as slow- and fast-adapting, similar to the regular type reported in the neocortex. The somata of the claustral neurons resembled those of cortical layer VIb cells characterized by diversity. In addition, some of the dendrites of these extended toward the superficial layers of the ventrolateral and lateral orbital cortex (VLO/LO), and arborized profusely in the claustral partition and the VLO/LO. Furthermore, some single neu- rons of the claustrum were shown to send their stem axons and collaterals to the VLO/LO, the agranular motor cortex (AgMCx) and other cortical areas by double staining with fluorescent dyes and intracellular staining. These findings strongly suggest that the rostral claustrum is a part of the layer VI of the adjacent cerebral cortex, i.e., VLO/LO, shares input to the VLO/LO in addition to input to the claustrum itself, and integrates these two inputs. Considering the gelatino- orbital projection and connections among the claustrum, VLO/ LO and AgMCx, the rostral claustrum may be an important region that integrates the affective-motivational aspects of pain and behavior.

(7) Synaptic connections and synchronized firing of cortical interneurons

Yasuo Kawaguchi, Satoru Kondo (National Institute for Physiological Sciences)
Yoshiyuki Kubota (Bio-Mimetic Control Research Center, RIKEN)

GABAergic interneurons in the rat frontal cortex are divided into 2 groups according to the intrinsic firing pattern: FS (fast-spiking) cells and non-FS cells. FS cells show abrupt episodes of nonadapting repetitive discharges of short-duration spikes and are immunoreactive for parvalbumin. Non-FS cells are further classified on the basis of neuropeptide and calretinin content. Somatostatin cells include Martinotti cells with ascending axonal arbors, innervating thin dendritic branches or spines. VIP and/or calretinin cells include double bouquet cells with descending axonal arbors innervating dendrites. Large CCK cells have axonal arbors making mul- tiple boutons on other cell bodies. In addition to intracortical synaptic connections, modulatory subcortical inputs may be also different among the GABA cell subtypes.Carbachol or muscarine affects the activities of peptide-containing GABAergic cells more than parvalbumin FS cells. Somatostatin or VIP cells are depolarized with spike firing, whereas large CCK basket cells are hyperpolarized.Continuous application of carbachol or muscarine induces two patterns of the increases of inhibitory postsynaptic currents in cortical cells: continuous and periodic ones.The periodical increase induced by carbachol or muscarine was synchronized among cortical cells. A few nonpyramidal cells showed the slow periodical discharges in response to carbachol. Cholinergic afferents from the basal forebrain may affect cortical activities through GABA cell subtypes differentially.

(8) Electrical Synapses and Networks of GABAergic Neurons in the Neocortex

Shaul Hestrin & Mario Galarreta (Stanford University, USA)

It has been suggested that GABAergic axo/dendritic and axo/somatic interactions among inhibitory neurons are critical in coordinating cortical activity. However, synaptic interactionsamong GABAergic cells and their relation to coordinated activity are only poorly understood. We have addressed these issues by recording simultaneously from pairs of fast-spiking (FS) cells, a kind of GABAergic neocortical interneurons. The FS cells were immunoreactive to parvalbumin and had local and horizontal axon projections making inhibitory contacts with both pyramidal neurons as well as other inhibitory neurons.We have found that in addition to GABAergic chemical synapses FS cells were electrically coupled. Electrical synapses appear to be cell-type specific. We have not found evidence of electrical coupling among pairs of neighboring pyramidal neurons. Moreover, we have not found electrical synapses between FS cells and other types of neocortical cells. When both chemical and electrical transmission were present between two FS cells, a presynaptic spike generated a dual component response in the postsynaptic cell. The peak of the electrical synapse mediated response occurred earlier than that of the GABAA-receptor mediated IPSP. We found that the efficacy of signal transmission through electrical synapse is frequency-dependent. Thus, we have directly demonstrated extensive electrical coupling among neocortical FS cells. The high degree of connectivity could establish a functional network of specific type of neurons within the neocortex. Given that inhibitory neurons control other neurons these networks could organize cortical activity and promote synchrony.

(9) Local circuit and representation of sound

Wen-Jie Song (Dept of Electronic Engineering, Osaka University)
Hideo Kawaguchi (Advanced Research Laboratory, Hitachi Ltd.)

Sound is decomposed in the cochlea into different fre- quency components. Each frequency component is detected by hair cells at a specific point of the basilar membrane. Components of lower frequencies are represented by the basilar membrane region close to the base of the cochlea, and components of higher frequencies are represented at regions close to the apex. Such spatial representation of sound frequency is often referred to as tonotopy. Tonotopy is maintained all through the central auditory pathway up to the cortex. In the cortex, neurons of the same characteristic frequency tend to stay together, along a dorso-ventral axis, forming an isofrequency stripe. Traditionally, isofrequency stripes in the cortex have been thought to be formed due to topographic projection from the thalamus, along the frequency axis and along the isofrequency dimension. In our report, we show evidence that cortical local circuits contribute signifi- cantly to the formation of isofrequency stripes.

(10) Classification of neuron types based on the firing patterns recorded in behaving monkeys

Akichika Mikami, Keichiro Kato, Shunpei Unno (Primate Research Institute, Kyoto University)
Youngnam Kang (School of Medicine, Kyoto University, Health Sciences University of Hokkaido)

In order to develop a new approach to classify neuron types applicable for the data taken in behaving monkeys, we measured the amplitudes of spikes, the duration of spikes, the inter-spike intervals and number of spikes within the bursting activities recorded in the frontal cortex of behaving monkeys. Following the results of the cluster analysis we could identify fast spiking (FS), fast rhythmic bursting (FRB) and intrinsic bursting (IB) neurons. FS neurons were characterized by shorter and relatively constant ISI (<5ms) and larger number of spikes (>6 spikes, often >10 spikes). FRB neurons were characterized by shorter and relatively constant ISI (<5ms) and smaller number of spikes (<6 spikes, usually 2 or 3 spikes) within a burst. In addition, FRB neurons often showed 20-80 Hz fast rhythmic burst, which is consistent with the characteristics of FRB neurons reported previous studies. IB neurons were characterize by longer and variable ISI (fluctuating between 4-12 ms) and smaller number of spikes (<6 spikes, usually 2-4 spikes) within a burst. The data suggested the possibility to use this approach to analyses neuronal circuits related the behavior.

(11) Velocity and Position Guided Orienting in Unrestrained Cats

Sasaki S., Yoshimura, K., Naito, K. (Dept. of Neurophysiology, Tokyo Metropolitan Institute for Neuroscience)

Moving and stationary visual stimulus may lead to elicit different types of orienting, because the moving visual stimulus activates retinal neurons sequentially along the trajectory of the moving stimulus, while the stationary stimulus activate neurons in a restricted area on which the the object projected on. We, thus, attempted to chara- cterized the differences of orienting elicited by moving and stationary stimuli. Cats were trained to stand in a small box and to fixate at a light spot projected on a center of the panel and were required to orient to the light spot which jumped from the fixation point to a target or moved with various speed in a ramp-hold manner. When a light spot jumped from a fixation point to a target, cats first moved their heads slowly toward the target with concomitant controversive eye movements due to the vestibulo-ocular reflex, resulting in a flat gaze traces and then made rapid head movements and saccades toward the target. Latencies of head movements were 100-200 ms. Switching from jumping to moving visual stimuli produced pronounced shortening of latencies of orienting and increases of head and gaze velocities. These results suggesting that there are two types of orienting, i.e., position guided and velocity guided orienting, which utilize position and velocity information of visual stimuli, respectively.

(12) Dynamic regulation of signal transmission in the local circuit of the superior colliculus

Yasuhiko Saito & Tadashi Isa (Dept. Integrative Physiology, National Institute for Physiological Sciences)

To understand the mechanisms of generation of motor commands in the superior colliculus (SC), we investigated activation properties of neurons in the intermediate layer (SGI) using whole-cell patch clamp technique in the SC slices obtained from 17- to 22-day old rats. In the presence of bicuculline (Bic, 10 mM), a single shock stimulation of the optic tract (OT) often induced the long-lasting depolarization with repetitive firings in SGI neurons. Moreover, when a local stimulation was applied in a small rectangle piece of slice isolated from the SGI, the SGI neurons in the rectangle exhibited the repetitive firings, suggesting the existence of neural elements within the SGI, which yielded bursting properties when GABAergic inhibition was removed. To further investigate the structure of local excitatory connec- tions in the SGI, we performed simultaneous recordings from a pair of SGI neurons. Application of Bic in addition to reducing extracellular Mg²⁺ induced synchronous spontaneous depolarization (> 20 mV) of neurons in the SGI frequently even when the two SGI neurons did not show the direct synaptic connections. All these results suggest that the local excitatory connections of the SGI neurons may be important in generation of the bursting motor command in the SGI.

(13) Mutual inhibition in the premotor circuit controlling saccade

Yoshida, K., Iwamoto, Y., Chimoto, S. and Shimazu, H. (Department of Physiology, Institute of
Basic Medical Sciences, University of Tsukuba, and CREST of JST)

The brain stem neural circuit that generates saccadic eye movements contains two essential classes of neurons, burst neurons(BNs) and omnipause neurons (OPNs).Since OPNs make direct inhibitory connections with BNs, it is believed that the duration of the BNs discharge is controlled by a pause of OPNs. Intracellular recordings were made from OPNs in alert cats to elucidate postsynaptic events during saccades and thereby determine what patterns ofafferent discharge impinge upon OPNs to cause their pause. The membrane potential of OPNs showed steep hyperpolarization in association with each saccade.The results indicate that the pause of activity in OPNs is initiated by an abrupt inhibition and maintained till the end of the saccade by afferents encoding eye velocity signals. We suggest that the eye velocity-related afferents originate from BNs and that powerful mutual inhibitory interactions between BNs and OPNs generate their comple- mentary discharge patterns.

(14) Synchronous oscillatory activity of local field potentials and neurons
in MI and SMA during bimanual movements of monkeys

Takashi Kawashima (Dept. Neurophysiol. Inst. of Equilibrium Res., Gifu Univ. Sch. of Med &
Dept physiol. and Biophys. and Regional Primate Res. Ctr., Univ. of Washington)
Thomas A. Knight, Eberhard E. Fetz (Dept physiol. and Biophys. and Regional Primate Res. Ctr.,
Univ. of Washington)

Simultaneous local field potential (LFP) were recorded in the right and left primary cortex (M1) and supplementary motor cortex (SMA) and parietal cortex (Area7) of two macaca nemestrina performing visually guided bimanual tracking task. All LFPs showed oscillation at 20-40Hz that could become bilaterally synchronous during the bimanual movements. Averaged cross-correlation coefficient (ACCC) between the recording sites were compared in three behav- ioral states: 1) pre-movement periods, 2) during the bimanual tracking periods and 3)reward periods. Regarding the hemi lateral connection, the values of ACCC were arranged in order SMA-Area7 > SMA-MI > MI-Area7 in both hemispheres in any of these behavioral periods. Regarding the bilateral connection, no significant changes of ACCC were observed in SMA and MI between pre-movement and the bimanual tracking periods. In the reward period the coefficient values were highest in any recording sites. In addition to the ordinary averaging method, time course analysis of cross correlation of LFPs were also executed.Bilateral oscillatory synchronous activities of LFPs were observed in M1 and SMA in accor- dance with a rapid initiation or cessation of bimanual move- ments.

(15) A new bipedally walking monkey model for the study of human bipedal locomotion

Shigemi Mori, Futoshi Mori, Katsumi Nakajima, Atsumichi Tachibana, Masahiro Mori,
and Chijiko Takasu (Department of Biological Control System, National Institute for Physiological Sciences)

To advance understanding of the CNS mechanisms in the human which control and integrate posture and bipedal locomotion, it is desirable to establish a non-human primate model.With such a model, it is possible to apply the state- of-the-art techniques of modern neuroscience to the problem of developing testable hypotheses on relevant CNS control mechanisms. By use of long-term operant conditioning, we have recently trained the normally quadrupedal young Japa- nese monkey (M. fuscata; estimated age; 3 to 4 years old) to stand upright and walk bipedally on a moving treadmill belt. After 3 to 4 months of regular locomotor training, they acquired a novel strategy of walking bipedally. With continuation of the locomotor learning together with monkey's physical growth over time, the monkeys refined gradually their walking patterns with less fluctuation of body axis. We have already shown that the kinematic features of our model's bipedal treadmill walking resemble those used in human locomotion. We have also found that bipedally walking monkeys can clear the obstacles attached on the moving treadmill belt with the larger flexion of hip and ankle joints than that observed during treadmill locomotion without an obstacle. When the monkey failed to clear the obstacle and stumbled, it adopted a defensive posture with rapid extension of the forelimbs forward and downward with lowering of the center of body mass to the treadmill surface. Our result indicates that "integrated" interactions of multiple body segments such as head, body, forelimbs and hindlimbs are necessary for preventing "stumbling" from "falling". All these results suggest that the monkey walking bipedally on a moving treadmill belt acquireda hip-knee strategy of recruiting and integrating both anticipa- tory and reactive neural mechanisms in the CNS to accomo- date its posture and locomotion to the external perturbations. Our new model monkey seems to employ CNS mechanisms similar to those in human subjects. With the use of non- invasive method such as positron emission tomography (PET), we are now exploring the potential CNS mechanisms and operating principles for the elaboration of monkey's bipedal locomotion with an upright posture.

(16) Selective expression of Ca²⁺-permeable ampa receptors in horizontal cells
in the superficial layer of rat superior colliculus

Toshiaki Endo, Tadashi Isa (Department of Integrative Physiology,
National Institute for Physiological Sciences)

Rectification property and Ca²⁺-permeability of AMPA- type glutamate receptors were investigated in six morpho- logically identified subclasses of neurons in the superficial layer of the superior colliculus (sSC) by whole-cell patch clamp recording technique in slice preparations obtained from rats (PND17-23). Both outwardly rectifying and inwardlyrectifying current responses were observed to pressure application of kainate (KA). Theses currents were completely abolished by 100 mM GYKI 52466, indicating that the KA-induced currents were mediated by AMPA-type glutamate receptors. The inwardly rectifying KA responses were suppressed by 1 mM spermine. The degree of inward rectification was inversely correlated with the reversal potential of KA responses in the isotonic Na+-free, high Ca²⁺ solution, indicating Ca²⁺-permeability was higher in inwardly rectifying AMPA receptors. The inwardly rectifying and Ca²⁺-permeable AMPA receptors were observed in a large proportion of horizontal cells with extensive dendritic arborization in the tangential direction. These results suggest that Ca²⁺-permeable AMPA receptors have specific role in regulation of lateral interaction in the visual field map in the sSC.

(17) Burst firing in layer III pyramidal neurons of cat sensorimotor cortex

Yoshihiro Nishimura, Hiroshi Shibuya, Toshihiro Asahara and Tetsuro Yamamoto (Department
of Physiology, Faculty of Medicine, Mie University)

The ionic mechanisms underlying burst firing in layer III neurons from cat sensorimotor cortex were examined by intracellular recording in a brain slice. Regular spiking was observed in 77.4 % of 137 neurons in response to constant intracellular current pulses. The rest of the neurons showed burst firing. An initial burst followed by regular-spike firing was observed in 71.0 % of 31 bursting neurons. The rest of the bursting neurons (N= 9) showed repetitive bursting. In the bursting neurons, spikes were triggered from the afterdepolarization (ADP) of the 1st spike of burst. The ADP was enhanced by a blockade Ca²⁺ channel. This enhanced ADP by Ca²⁺ channel blockade was apparent after blockade of the afterhyperpolarization by apamin or intracellular Ca²⁺ chelation by EGTA. These agents switched the regular spiking pattern into a bursting one, but could not change a burst firing into a regular spiking. Four neurons responding with a single initial burst in control solution responded with repetitive bursting after application of these agents. The ADP was blocked and the burst firing changed to regular spiking by tetrodotoxin (TTX) or intracellularly applied QX314. We concluded that a TTX- and QX314- sensitive Na+ current underlies the ADP and contributes to the burst firing of layer III neurons from the cat cortex.

(18) Interaction of horizontal and vertical inputs to pyramidal cells in the superficial layers
of the cat visual cortex

Hiromichi Sato (School of Health and sport Sciences, Osaka University)
Yumiko Yoshimura (Department of Neuroscience, Osaka Bioscience Institute & Department of Visual Neuroscience, Research Institute of Environmental Medicine, Nagoya University)
Kazuyuki Imamur and Yasuyoshi Watanabe (Department of Neuroscience, Osaka Bioscience Institute)

The purpose of this study is to elucidate the integrative input mechanisms of pyramidal cells receiving horizontally projecting axon collaterals (horizontal projection) and vertical input from layer IV.We performed whole-cell recordings from pyramidal cells in layer II/III and focally activated other single pyramidal cells monosynaptically connected via long- distance horizontal(LH) projections (the distance between pre- and post-synaptic cells was350-1200μm) in slice prepa- rations of the kitten primary visual cortex.In addition, presynaptic single fibers in layer IV (vertical input) and/or short-distance horizontal (SH) inputs from neighboring single pyramidal cells (distance within 100μm) in layer II/III were activated. Unitary excitatory postsynaptic potentials (EPSPs) evoked by the activation of LH and SH connections had smaller amplitude and larger coefficient of variation than those evoked by stimulating the vertical input. Paired-pulse stimulation of the LH and SH inputs caused the depression of the second EPSP, whereas that of vertical inputs caused either facilitation or depression of the second EPSP. The EPSPs evoked by simultaneous activation of LH and vertical inputs summated linearly at the resting membrane potential.However, the EPSPs evoked by stimulation of the two inputs were nonlinearly (supralinearly) summated when the postsynaptic membrane was depolarized to a certain level. Similar EPSP interaction was observed in response to simulta- neous activation of the LH and SH inputs.

(19) Spatiotemporal interaction of response to multiwhisker stimulation in the barrel cortex neurons of rats

Satoshi Shimegi, Takafumi Akasaki, Hiromichi Sato (School of Health and sport Sciences, Osaka University)

To understand the physiological properties and anatomical organization of the spatiotemporal interaction of the responses to multiwhisker stimulation in neurons of the rat barrel cortex, single-unit recordings of 114 neurons were performed across all layers (layer II/III, n =39 ; IV, n = 33 ; V/VI, n = 42) of the barrel cortex of anesthetized rats. Two neighboring principal and adjacent whiskers (PW and AW, respectively) in the same row were deflected rostrally or caudally at varying interstimulus intervals (ISIs).In 37 % of the cells tested, multiwhisker stimulation exhibited response facilitation; the response to the combined stimulus was larger than the sum of the responses to stimulation of the individual whiskers. Among them, 75 % and 60 % were selective for the combination of the PW with a particular AW and for a particular direction of whisker deflec- tion, respectively. Most cases of facilitation were observed at short ISIs (<4 msec). Multiwhisker stimulation at longer ISIs (>8 msec) caused a suppression of the response to the second whisker stimulation.Facilitation was evoked predomi- nantly in layer II/III cells (69 %) and most of them were located close to the border between two columns, where strong excitatory inputs from the adjacent column were expected. Our results suggest that the spatiotemporal patterns of multiwhisker stimulation, such as whisker combination, direction of deflection and timing, are expressed as different pattern and magnitudes of response interaction, which depends on the location of the recorded cell including proximity of cells to home and adjacent barrel columns and lamination.

(20) Functional Development of the Recurrent Inhibitory Network of the Mauthner Cell in Zebrafish larvae

Masaharu Takahashi (Lab. of BrainScience, Div. of Biophysical Engineering, Grad. Sch.
of Engineering Science, Osaka Univ., JSPS Research Fellow)
Madoka Narushima, Yoshinnobu Murayama (Lab. of BrainScience, Div. of Biophysical Engineering,
Grad. Sch. of Engineering Science, Osaka Univ.)
Yoichi Oda (Lab. of BrainScience, Div. of Biophysical Engineering, Grad. Sch. of Engineering Science,
Osaka Univ., and PRESTO 21)

Output of neuronal activity is restrained by the local network. The Mauthner (M-) Cell, a large reticulospinal neuron in the hindbrain, generates a single action potential that initiates the fast escape behavior of teleost fish from a sudden aversive stimulus. Activation of the single action potential of the M-cell in the adult goldfish is controlled, at least in part, by recurrent inhibition of the M-cell mediated through glycinergic interneurons. In the present study, we examined when the feedback circuit is established, with using confocal Ca imaging of the M-cell in the intact zebrafish larvae. The M-cells were retrogradly labeled with a fluorescent calcium indicator, Calcium Green Dextran, injected at the spinal cord of zebrafish 4-16 days post fertilization (dpf). Stimulation of the spinal cord induced increase in fluorescence intensity in all-or-none manner, indicating antidromic (AD)activation of the M-cell. The intensity of the Ca response was reduced (88.6±3.3%, n=19) when the second AD spike was evoked at 5ms after the first spike during the period of recurrent inhibition.Applying a glycine antagonist, strychnine, blocked the reduction of Ca response. Thus, the reduction of Ca response observed in zebrafish 4-16dpf indicates the shunt of AD spike produced by the inhibitory conductance of the M- cell recurrent network. These results suggest that recurrent inhibition of the M-cell becomes functional in zebrafish larvae before 4dpf.

(21) Fluorescence imaging of the protein kinase C translocation
in the hippocampal neurons

H. Tsubokawa and S. Takagi (National Institute for Physiological Sciences)

In neurons of the CNS, little is known about spatial and temporal profiles of the PKC activation in relation to neuronal activities. In this study, we tried to detect translocation of the PKC, which is followed by binding to the substrates. A com- bination of intracellular recording and fluorescence imaging was applied to CA1 pyramidal neurons of mouse hippocampal slices. Cells were loaded with 50 μM fim-1, PKC-binding dye, through a patch-pipette. Bath application of 100 nM phorbol 12-myristate 13-acetate, an activator of the PKC, reversibly changed the spatial pattern of the fim-1 fluorescence.The fim-1 fluorescence transiently decreased, then a bright area appeared around the cell membrane regions within 15 min. During this period, the amplitude of EPSCs induced by stimulation of Shaffer collateral / commissural fibers increased, and the paired pulse facilitation of the EPSCs was reduced. Train of action potentials induced similar transient decreases in the fim-1 fluorescence with increases in [Ca²⁺]_i. In the presence of PKC inhibitors, pharmacological or electrical stimulation did not induce significant changes in the spatial patterns of the fim-1 fluorescence without affecting changes in [Ca²⁺]_i. These results suggest that optical imaging of fim-1 fluorescence is useful for detecting activation of the PKC in specified neurons in the brain slice preparation.

(22) Calciumu-sensitive cation current accompanying activation of
nicotinic acetylcholine receptors in rat mesopontine dopamine neurons

Tetsuji Yamashita, Tadashi Isa (Department of Integrative Physiology,
National Institute for Physiological Sciences)

The mesopontine dopamine neurons receive cholinergic inputs from the pedunculopontoine nucleus as a main excitatory input. We analyzed the current responses of dopamine neurons to the puff application of acetylcholine (ACh;1mM) using whole cell patch clamp recording in the midbrain slices obtained from rats (PND12-17) and clarified three points as follows; 1. The puff application of ACh induced fast inward current which was suppressed by mecamylamine, suggesting that the current was mediated by nicotinic ACh receptors (nAChRS). 2. The ACh- induced current was partially suppressed by fulfenamic acid (FFA; 200 mM), an antagonist of Ca²⁺-activated nonselective cation channels (CAN channels). However, FFA did not sup- press the ACh-induced current in the Ca²⁺-free extracellular solution. These results suggested that we could exclude the possibility FFA suppressed the nAChR-mediated current itself, but instead FFA suppressed the Ca²⁺-dependent current secondarily activated by the nAChR-mediated current. 3. Suppression of the ACh-induced current by FFA could be observed under the presence of thapsigargin (1mM), suggesting that the Ca²⁺ influx through the nAChRs could directly activate the FFA-sensitive current, presumably mediated by CAN channels.We have concluded that the ACh-induced current in the dopamine neurons contains FFA-sensitive Ca²⁺ depen- dent current that is subsequently activated by the Ca²⁺ influx through the nAChRs.



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