DEPARTMENT OF DEVELOPMENTAL PHYSIOLOGY

Outline

Department of Developmental Physiology was founded in 2003 with 3 full-staff divisions and 1 adjunct division, for the purpose of clarifying the physiological mechanism of mental and physical development of human beings. One of the divisions was transferred from the previous Higher Brain Function Project, Department of Integrative Physiology, headed by Prof. Tadashi Isa. This division (Division of Behavioral Development) studies the development and post-injury recovery of the neural systems controlling the eye and hand movements. Prof. Junichi Nabekura was elected as a chair of the Division of Homeostatic Development and has initiated the activity of the division since 2003. This division studies the development of synapses in the central nervous system and remodeling of neural circuits after the brain injury by using electrophysiological techniques and imaging with multi-photon microscopes. Prof. Yasuhiko Minokoshi was elected as the chair of Division of Reproductive/Endocrine Development, and started researches on neural control of metabolism, especially focused on the function of the hypothalamus, to obtain better understanding of the molecular mechanisms of pathophysiology of obesity and diabetis mellitus. Division of Adaptation Development, an adjunct division, had been chaired by Prof. Toshihiko Yada of Jichi Medical University, whose is a specialist on the energy and glucose homeostasis, since 2009.

We are investigating the neural systems controlling saccadic eye movements and grasping hand movements. We analyze the structure and function of local circuits and large-scaled networks involved in these motor systems. We are also interested in plastic compensatory mechanism following lesion of related structures as detailed below;

1. Saccadic eye movements
(1)Electrophysiological analysis of local circuits of the superior colliculus (SC), a pivotal midbrain center for saccade control, by using in vitro slice preparation.
(2)Analysis of local circuit of SC and large scaled network involving the cerebral cortex for saccade generation in anesthetized animals (rodents and non-human primates).
(3)Molecular mechanism of saccade generator circuits by using genetically manipulated mice.
(4)Analysis of dynamic properties of saccade-related circuits by applying electrophysiological and pharmacological techniques in awake behaving non-human primates.
(5)To clarify the neural mechanism of saccade control and visual awareness in "blindsight" patients, we are analyzing the saccadic behaviors and neuronal activities in macaque monkeys with unilateral lesion of the primary visual cortex (V1), as an animal model of "blindsight".

2. Dexterous hand movements
We clarified the existence of oligosynaptic (indirect) pathway from the primary motor cortex to hand motoneurons mediated by interneurons in the cervical spinal cord. Moreover, we observed the behavior of the monkeys in which direct cortico-motoneuronal connections are transected while the indirect pathway remained intact and found that the monkeys can perform precision grip after 1-3 months of recovery period. To explore the basic mechanism involved in the compensatory mechanisms, we use multidisciplinary approaches including electrophysiology, non-invasive brain imaging with positron emission tomography (PET) and analysis of gene expression by DNA microarray and in-situ hybridization.

Recently we start to approach the analysis of neural systems controlling movements by the manipulation of the function. One is the Brain-machine Interface (BMI). This technology with decoding neural activity would be useful for the analysis of neural system. The other is the gene transfer using virus vector. For example, the optogenetics is a high-powered tool to discriminate the neurons with particular function or connections.

Figure 1. Schematic of local circuit underlying GABA_BR-mediated regulation of bursts in the SC. Postsynaptic GABA_BRs expressed both in NFV and WFV cells and presynaptic GABA_BRs located on gluamatergic synaptic terminals in the SGS are activated by synaptically released GABA during bursts of SGS GABAergic neurons. Hyperpolarization in NFV cells, shunting inhibition in WFV cells, and reduction of glutamate release may contribute to the limitation of burst duration. When GABA_BRs are blocked, burst duration in the SGS may be prolonged in an NMDAR-dependent manner, and then, the prolonged burst may spread to the SGI. Thus the burst duration of the SGI may also be prolonged.

Figure 2. (a) The schematic drawing indicates the cutting plane of horizontal slice of superficial SC with arrangement of stimulating and recording system from the coronal view. The red arrows show directions of electrical stimulation that were applied. (b) Top view of the horizontal slice of superficial SC overlying an electrode array. The pink asterisk indicates the location of recorded neuron. Electrical stimulationwas applied from each of six electrodes in the white square while recording from the neuron. (c) The evoked EPSPs or IPSPs from the stimulation at electrode number 1-6 in figure b are shown for single pulse (top row blue traces) and 200 Hz (bottom row red traces) stimulation. (d) The integration of EPSP (positive value) and IPSP (negative value) from the data in panel c during 0-50 ms after stimulation onset were summarized and plotted on the distance axis that the closest electrode to the cell was set to be zero. Percent of maximal responses were plotted on the distance between the stimulating electrode to the cell closest electrode forsingle pulse (blue) and 11 pulses/ 200 Hz (red).

Figure 3. Neuronal activities in the SC ipsilateral to V1 lesion correlated to the monkey's performance. When the monkey succeed to detect them ('Hit' trial), this neuron exhibited clear visual responses to the visual stimuli presented in the visual field affected by the lesion, while when the monkey failed to detect them ('Miss' trial), these responses attenuated although the same visual stimuli were presented.

Figure 4. After spinal cord injury, a subcortical oscillator commonly recruits hand/arm muscles, via remaining pathways such as reticulospinal and/or propriospinal tracts, independent of cortical oscillation, and contributes to functional recovery.

Staff

In the last stage of neural development, a massive re-arrangement of neuronal circuits takes place. This is associated with an alteration of functional neural circuits, resulting in the changes in various brain functions (e.g. behavior, sensory function and biological rhythm). Our research aim is to understand the developmental re-arrangement of brain function at neuronal circuits and synaptic levels by using various approaches, e.g. in vivo imaging with advanced two photon microscopy, electrophysiology and molecular biological techniques. We also focus on the re-appearance of immature characteristics at the molecular, synapse and circuit levels after acute brain damage.

To achieve cutting edge technique for in vivo imaging with two photon microscopy, we have improved the laser light path and the cranial attachment for imaging. We succeeded in achieving a distinct imaging of various fine structures through entire layers of mouse cortex.

(1) By employing this technique, we attempt to elucidate the mobility of neuronal spines and various glia during development and after neuronal injury. We visualized the microglia surveillance for synapses in in vivo. In the intact brain, microglia attached onto the boutons, presynaptic structures, and dendritic spines, postsynaptic structures, for 5 min every 1 hour, exactly. In the damaged brain, this contact became much prolonged to more than 1 hour in duration. Occasionally, the damaged synapses were eliminated after the prolonged contact. Microglia seemed to disconnect the severely damaged neuronal circuits. In addition, the rate of synapse turnover increased at the hemisphere contralateral to damaged somatosensory cortex during limited period after the infarction, resulting in generating new circuits compensating the loss of function. In addition, we also focus on the remodeling of neuronal circuits in case of chronic pain model and GABAergic migration in the immature cortex.

(2) In immature animals, GABA induces neuronal depolarization and often acts as an excitatory substance. Intracellular CI^- concentration decreases during development, resulted from developmental switch of intracellular CI^- regulators, e.g. K⁺ CI^- cotransporter2 (KCC2) and Na⁺, K⁺, CI^- cotransporter 1 (NKCC1). Phosphorylation of KCC2 induces its clustering at the lipid raftand apprears its function. Neuronal damage induced dephosphorylation of KCC2, resulting in an increase in the intracellular Cl^- concentration and switching GABA response from hyperpolarization to depolarization. We also study functional relevance of GABA excitation in Gn-Rh neuron, which expressed low KCC2 even in the mature, by employing several gene-manipulated mice.

(3) We reported a new form of synapse development. Transmitters to the lateral olive neurons, auditory relay neurons, switch from GABA in the immature to glycine in the mature. The transmitter switch proceeds at single synaptic terminal. This could be one of unique synaptic plasticity in developing neural circuits. We now focus on elucidating underlying mechanisms, e.g. related trophic factors.

(4) we are interested in studying the modulatory action of various nave and natural bioactive substances, e.g., TRH on the synaptic transmission and neuronal excitability by performing patch clamp recording on slice and from acutely dissociated neuron.

Staff

The animal body has an integrated-regulatory system for "homeostasis" that maintains a normal, constant-internal state by responding to changes in both the external and internal environments. Within the central nervous system, the hypothalamus is a crucial center that regulates the homeostatic activities by integrating autonomic nervous system, endocrine system and immune function. In this division, we are extensively investigating the role of hypothalamus in body energy balance in mammals. These studies are now important for better understanding the molecular mechanisms behind pathophysiology of obesity and diabetes mellitus. The main subjects of our current research are as follows:

(1) Molecular mechanism of the hypothalamic regulation of food intake.

(2) Regulatory role of the hypothalamic-sympathetic nervous system in glucose and lipid metabolism.

(3) Signaling pathway for metabolic action of leptin and adipokines in peripheral tissues.

(4) Role of AMPK and AMPK-related family in the regulation of metabolism in physiological and pathophysiological conditions.

Leptin controls body energy metabolism by reciprocally regulating AMP kinase in the hypothalamus and skeletal muscle
Leptin activates AMP kinase (AMPK) in skeletal muscle directly at the muscle level and indirectly through the hypothalamic-sympathetic nervous system. Leptin also inhibits food intake by suppressing AMPK activity in the hypothalamus. Reciprocal regualtion of AMPK activity in the hypothalamus and skeletal muscle is necessary for the leptin's effect on energy metabolism. We are intensively studying the molecular mechanism for the reciprocal regulation of AMPK activity in the hypothalamus and skeletal muscle.

Regulatory role of the hypothalamic nuclei in glucose metabolism in peripheral tissues in response to leptin
Leptin activates POMC neurons in arcuate hypothalamus (ARC) via VMH neurons, thereby stimulating melanocortin receptor (MCR) in VMH and PVH neurons. Activation of MCR in VMH stimualtes gluocse uptake in BAT, heart and skeletal muscle, while MCR in PVH stimulates glucose uptake in BAT preferentially.

Stimulatory role of orexin in glucose metabolism in skeletal muscle
Orexins are hypothalamic neuropeptides that play important roles in the regulation of sleep/wakefulness, feeding and motivated behavior. We found that orexin neurons activate in reponse to tasting and anticipation of feeding, and then activate VMH neurons. Actiavated VMH neurons stimulate glucose utilizaiton in skeletal muscle preferentially, thereby preventing diet-induecd hyperglycemia.

Staff

Regulation of insulin release: 1) Elucidating mechanisms by which ghrelin inhibits insulin release, and establishing the basis for treating type 2 diabetes with ghrelin blockade that promotes insulin release. 2) Elucidating the role of Kv channels of islet b-cells in regulation of insulin release.

Regulation of feeding: 1) Elucidating feeding-regulating neural pathway in the hypothalamus, particularly that from the 1^st order center Arcuate nucleus to the integrative center Paraventricular nucleus. 2) Elucidating mechanisms how novel anorectic peptide Nesfatin-1 and classical peptide Oxytocin regulate feeding and exploring their (patho)physiological roles and therapeutic potential. 3) Interplay of brain and peripheral organs in regulation of feeding and body's homeostasis.

Fig 1. Mechanism for inhibition of insulin secretion by ghrelin. (Dezaki K., Yada T. et al.: Diabetes 56: 2319-2327, 2007; Dezaki K., Sone H., Yada T.: Pharmacol. Ther. 118: 239-249, 2008)

Fig 2. Endogenous ghrelin in islets inhibits and its blockade promotes insulin secretion. (Dezaki K., Yada T. et al.: Diabetes 53: 3142-3151, 2004; Diabetes 55: 3486-3493, 2006.; Yada T., Dezaki K., et al. Curr. Diab. Rev. 4: 18-23, 2008.)

Fig 3. Nesfatin-1-operative PVN oxytocinergic signaling to NTS POMC neurons causes melanocortin-dependent satiety
Central Nesf-1 or food intake activates Nesf-1 and Oxt neurons in PVN. In PVN, endogenous Nesf-1 serves as a paracrine/autocrine or local neuronal stimulator of Oxt neurons. PVN oxytocinergic signaling to NTS activates POMC neurons, inducing melanocortin-dependent satiety. (Maejima Y., Yada T. et al.: Cell Metabolism 10: 355-365, 2009)

Staff