TRPA1 as a O2 sensor detects microenvironmental hypoxia in the mice anterior cingulate cortex.

Transient receptor potential ankyrin 1 (TRPA1) is a member of the TRP channel family and is expressed in peripheral and central nervous systems. In the periphery, TRPA1 senses cold and pain. However, the functions of TRPA1 in the CNS are unclear. Here, we examined the roles of TRPA1 on neural activity and synaptic transmission in layer II/III pyramidal neurons from mice anterior cingulate cortex (ACC) by whole-cell patch-clamp recordings. The activation of Cinnamaldehyde (CA), which is TRPA1 agonist produced inward currents and these were blocked by the TRPA1 antagonists. Furthermore, activating TRPA1 changed the properties of action potentials such as the firing rate, rise time and decay time. In contrast, stimulating TRPA1 did not alter the spontaneous synaptic transmission. Finally, we examined the functional role of TRPA1 on neurons in a hypoxic environment. We induced an acute hypoxia by substituting nitrogen (N2) gas for oxygen (O2) in the external solution. N2 produced biphasic effects that consisting of inward currents in the early phase and outward currents in the late phase. Importantly, blocking TRPA1 reduced inward currents, but not outward currents. In contrast, a KATP channel blocker completely inhibited outward currents. These results suggest that TRPA1 acts on postsynaptic neurons in the ACC as an acute O2 sensor.

Eye movement activity in normal human fetuses between 24 and 39 weeks of gestation.

Rapid eye movement (REM) sleep occurs throughout a relatively large proportion of early development, and normal REM activity appears to be required for healthy brain development. The eye movements (EMs) observed during REM sleep are the most distinctive characteristics of this state. EMs are used as an index of neurological function postnatally, but no specific indices of EM activity exist for fetuses. We aimed to identify and characterize EM activity, particularly EM bursts suggestive of REM periods, in fetuses with a gestational age between 24 and 39 weeks. This cross-sectional study included 84 normal singleton pregnancies. Fetal EMs were monitored using real-time ultrasonography for 60 min and recorded as videos. The videos were manually converted into a time series of EM events, which were then analyzed by piecewise linear regression for various EM characteristics, including EM density, EM burst density, density of EMs in EM bursts, and continuous EM burst time. Two critical points for EM density, EM burst density, and density of EMs in EM bursts were evident at gestation weeks 28-29 and 36-37. Overall EM activity in human fetuses increased until 28-29 weeks of gestation, then again from 36-37 to 38-39 weeks of gestation. These findings may be useful for creating indices of fetal neurological function for prognostic purposes.

Glycine plays a crucial role as a co-agonist of NMDA receptors in the neuronal circuit generating body movements in rat fetuses.

Neuronal circuits generating fetal movements in mammals are localized in the brainstem and the spinal cord. It has been shown that glycine plays an important role through the strychnine-sensitive glycine receptors in these circuits. However, the role of glycine as the NMDA receptor co-agonist in fetal period is not fully understood. In this study, we examined the contribution of glycine to the perinatal rat spinal circuit generating forelimb movements utilizing isolated brainstem-cervical-spinal-cord preparations. In late embryonic-days-preparations, spontaneous motor bursts related to forelimb movements (forelimb-movement-related bursts; FMRBs) and respiration-related activity were observed. In neonatal preparations, spontaneous FMRBs were not observed but periodic motor bursts resembling the FMRBs could be induced after bath application of strychnine (strychnine-induced motor bursts; SIMBs). Both FMRBs and SIMBs were blocked by either the NMDA receptor antagonist APV or the antagonists of the glycine binding site of NMDA receptors [5,7-dichlorokynurenic acid (DCKA) or L-689560]. Furthermore, these motor bursts were facilitated by the glycine uptake blocker sarcosine. This effect of sarcosine was blocked by DCKA. The findings indicate that glycine plays a crucial role as a NMDA receptor co-agonist in generating spontaneous fetal motor activity before functioning as a classical inhibitory neurotransmitter in suppressing the fetal neuronal circuits.

KCC2-mediated regulation of respiration-related rhythmic activity during postnatal development in mouse medulla oblongata.

GABA acts as inhibitory neurotransmitter in the adult central nervous system but as excitatory neurotransmitter during early postnatal development. This shift in GABA's action from excitation to inhibition is caused by a decrease in intracellular chloride concentration ([Cl(-)]i), which in turn is caused by changes in the relative expression levels of the K(+)-Cl(-) co-transporter (KCC2) and the Na(+), K(+)-2Cl(-) co-transporter (NKCC1) proteins. Previous studies have used slices containing the medullary pre-Bötzinger complex (pre-BötC) to record respiration-related rhythmic activity (RRA) from the hypoglossal nucleus (12 N). The role of GABAergic transmission in the regulation of medullary RRA neonatally, however, is yet to be determined. Here, we examined how GABA and chloride co-transporters contribute to RRA during development in the 12 N where inspiratory neurons reside. We recorded extracellular RRA in medullary slices obtained from postnatal day (P) 0-7 mice. RRA was induced by soaking slices in artificial cerebrospinal fluid (aCSF) containing 8mM-K(+). Application of GABA significantly increased the frequency of RRA after P3, whereas application of a KCC2 blocker (R (+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-indenyl-5-yl)oxy]acetic acid (DIOA)) significantly decreased the frequency of RRA after P1. In addition, dense KCC2 immunolabeling was seen in the superior longitudinalis (SL) of the 12 N, which is responsible for retraction of the tongue, from P0 and P7. These results indicate that GABA administration can increase RRA frequency during the first week following birth. This in turn suggests that decreasing [Cl(-)]i levels caused by increasing KCC2 levels in the 12 N could play important roles in regulating the frequency of RRA during development.

Orexin-neuromodulated cerebellar circuit controls redistribution of arterial blood flows for defense behavior in rabbits.

We investigated a unique microzone of the cerebellum located in folium-p (fp) of rabbit flocculus. In fp, Purkinje cells were potently excited by stimulation of the hypothalamus or mesencephalic periaqueductal gray, which induced defense reactions. Using multiple neuroscience techniques, we determined that this excitation was mediated via beaded axons of orexinergic hypothalamic neurons passing collaterals through the mesencephalic periaqueductal gray. Axonal tracing studies using DiI and biotinylated dextran amine evidenced the projection of fp Purkinje cells to the ventrolateral corner of the ipsilateral parabrachial nucleus (PBN). Because, in defense reactions, arterial blood flow has been known to redistribute from visceral organs to active muscles, we hypothesized that, via PBN, fp adaptively controls arterial blood flow redistribution under orexin-mediated neuromodulation that could occur in defense behavior. This hypothesis was supported by our finding that climbing fiber signals to fp Purkinje cells were elicited by stimulation of the aortic nerve, a high arterial blood pressure, or a high potassium concentration in muscles, all implying errors in the control of arterial blood flow. We further examined the arterial blood flow redistribution elicited by electric foot shock stimuli in awake, behaving rabbits. We found that systemic administration of an orexin antagonist attenuated the redistribution and that lesioning of fp caused an imbalance in the redistribution between active muscles and visceral organs. Lesioning of fp also diminished foot shock-induced increases in the mean arterial blood pressure. These results collectively support the hypothesis that the fp microcomplex adaptively controls defense reactions under orexin-mediated neuromodulation.

Imaging of neural ensemble for the retrieval of a learned behavioral program.

The encoding of long-term associative memories for learned behaviors is a fundamental brain function. Yet, how behavior is stably consolidated and retrieved in the vertebrate cortex is poorly understood. We trained zebrafish in aversive reinforcement learning and measured calcium signals across their entire brain during retrieval of the learned response. A discrete area of dorsal telencephalon that was inactive immediately after training became active the next day. Analysis of the identified area indicated that it was specific and essential for long-term memory retrieval and contained electrophysiological responses entrained to the learning stimulus. When the behavioral rule changed, a rapid spatial shift in the functional map across the telencephalon was observed. These results demonstrate that the retrieval of long-term memories for learned behaviors can be studied at the whole-brain scale in behaving zebrafish in vivo. Moreover, the findings indicate that consolidated memory traces can be rapidly modified during reinforcement learning.

Loss of pre-inspiratory neuron synchroneity in mice with DSCAM deficiency.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam (-/-)) died within 24 hours after birth. Whole body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Further, a medulla-spinal cord preparation of Dscam (-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory (Pre-I) neurons located in the rostral ventrolateral medulla (RVLM) and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam (-/-) mice. Dscam (+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam (-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner. These results have been published (Amano et al. 2009).

Central respiratory failure in a mouse model depends on the genetic background of the host.

To define the mechanisms of human congenital central respiratory failure, we are examining gene-deficient mice with central respiratory failure. However, the influence of the genetic background of the mice may play an important role in the phenotype of the mice. Therefore, we examined developmental respiratory adaptation in several mouse strains. Neonatal mice from P0 to P3 were examined by whole-body plethysmography and the electro- physiological analysis using brainstem-spinal cord preparations. Our results show that respiratory maturation becomes progressively fixed after birth and that the rate of progression depends on the genetic background of the mice. In particular, the progression of C57BL/6 mice was delayed compared to that of BALB/c mice.

Adrenaline modulates on the respiratory network development.

Adrenaline regulates respiratory network, however, adrenergic contribution to the developing respiratory center has not well studied. Adrenaline application on embryonic day 17 medulla-spinal cord block preparations abolished non-respiratory activity and enhanced respiratory frequency. Phentolamine application on neonatal brainstem-spinal cord preparations that produced stable neonatal respiration resulted in respiratory destabilization. In E19 rat, adrenaline switched from enhancement to depression of the respiratory rhythm. Adrenaline modulated GABAergic synaptic transmission to respiratory neurons in late developmental stage. These results suggest that the involvement of central adrenergic modulation on the respiratory network maturation.

Ontogeny of Cl- homeostasis in mouse hypoglossal nucleus.

We tested the immunoreactivity of KCC2 using KCC2 antibody in the developmental mouse medulla. Age-dependent changes in immunoreactivity were remarkable in the hypoglossal nucleus, and interestingly, the immunoreactivity in the hypoglossal nucleus relative to the dorsal vagal nucleus at P0 appeared to be higher than that of P7. Thus Cl(-) homeostasis in the hypoglossal nucleus might be differentially regulated in the developmental stage.

Respiratory activity of the neonatal dorsolateral pons in vitro.

The lateral and medial parabrachial and the Kölliker-Fuse nuclei (NPB/KF) are well known respiratory modulating centers in adulthood, but their role in neonates is largely unknown. We examined the role of the NPB/KF using hemi-sectioned pons-brainstem-spinal cord preparations in neonatal rats. Electrical stimulation applied at various intensities and delays in relation to the onset of spontaneous inspiratory C4 bursts, evoked transient depression or termination of C4 activity. This depression/termination was greatly attenuated either after perfusion of the NMDA-receptor antagonists (MK-801 or APV) or after microinjecting MK-801 into NPB/KF. Furthermore systemic application of the GABA-A receptor antagonist bicuculline reduced NPB/KF evoked inhibition of the C4 burst. Finally, we identified inspiratory, tonic inspiratory, expiratory, and inspiratory-expiratory (I-E) neurons which was major in the recorded neurons in the NPB/KF using the whole-cell patch-clamp method. MK-801 significantly decreased the driving potential and burst duration of I-E neurons. We conclude that neonatal NPB/KF mediated inspiratory off-switch operates on similar synaptic mechanisms as an adult.

DSCAM deficiency causes loss of pre-inspiratory neuron synchroneity and perinatal death.

Down syndrome cell adhesion molecule (DSCAM) is a neural adhesion molecule that plays diverse roles in neural development. We disrupted the Dscam locus in mice and found that the null mutants (Dscam(-/-)) died within 24 h after birth. Whole-body plethysmography showed irregular respiration and lower ventilatory response to hypercapnia in the null mutants. Furthermore, a medulla-spinal cord preparation of Dscam(-/-) mice showed that the C4 ventral root activity, which drives diaphragm contraction for inspiration, had an irregular rhythm with frequent apneas. Optical imaging of the preparation using voltage-sensitive dye revealed that the pre-inspiratory neurons located in the rostral ventrolateral medulla and belonging to the rhythm generator for respiration, lost their synchroneity in Dscam(-/-) mice. Dscam(+/-) mice, which survived to adulthood without any overt abnormalities, also showed irregular respiration but milder than Dscam(-/-) mice. These results suggest that DSCAM plays a critical role in central respiratory regulation in a dosage-dependent manner.

Adrenaline contributes to prenatal respiratory maturation in rat medulla-spinal cord preparation.

Adrenaline is a potent respiratory regulator. However, adrenergic contribution to the developing respiratory center has not been studied extensively. Adrenaline application on embryonic day 17 medulla-spinal cord block preparations abolished non-respiratory activity and enhanced respiratory frequency. Phentolamine application on neonatal blocks that produced stable neonatal respiration resulted in respiratory destabilization. These results suggest that central adrenergic modulation is involved in fetal respiratory development and maintenance of stable respiration.

Dopamine desynchronizes the pace-making neuronal activity of rat respiratory rhythm generation.

In an excised Wistar rat medulla-spinal cord block preparation we previously found that dopamine slows respiratory rhythm by activation of dopamine D(4) receptors [Fujii et al., (2004)Neurosci. Res., 50, 355-359.] In the present paper, we investigated the effect of dopamine on pre-inspiratory (Pre-I) and inspiratory (I) neurons using the combination of an optical recording technique with a voltage-sensitive dye, unit recording and patch-clamp recording. Optical imaging of the ventral surface of the block preparation disclosed different locations and activity patterns of Pre-I and I neurons. In addition to slowing the rhythm, dopamine depressed respiratory activity of Pre-I neurons collectively but not that of I neurons. The dopaminergic suppression of Pre-I neurons was mimicked by a dopamine D(4) receptor agonist, PD168077. Unit recording and patch-clamp recording demonstrated that dopamine depolarizes Pre-I neurons, disperses Pre-I firing and depresses Pre-I phase postsynaptic potentials (PSPs) of I neurons. Immunohistological investigation revealed that Pre-I neurons express dopamine D(4) receptors. We found that approximately 60% of Pre-I neurons express dopamine D(4) receptors. These results show that dopaminergic respiratory rhythm depression is due to dispersion of synchronized Pre-I driving of I neurons caused by dopamine D(4) receptor stimulation of Pre-I neurons.

Development of spontaneous mouth/tongue movement and related neural activity, and their repression in fetal mice lacking glutamate decarboxylase 67.

Spontaneous body movement starts at early fetal stage, at embryonic day (E) 12-15 in mice. In the present study, the movement of the head region was studied in E13-14 mice by in utero ultrasound imaging, together with the in vitro recording of underlying neural activities in the hypoglossal nerve and the ventral root of the upper cervical cord of an isolated brainstem-spinal cord preparation. The role of gamma-aminobutyric acid (GABA) in the generation of fetal movement was assessed using mice lacking GABA-synthesizing glutamate decarboxylase 67 (GAD67). At E14, mouth opening and tongue withdrawal were observed independently at frequency of 14/h each. This movement was rarely observed in the GAD67-deficient mouse. The intraventricular administration of picrotoxin or 3-mercaptopropionic acid abolished mouth opening in the wild-type mice. In a brainstem-spinal cord preparation, three types of neural discharge were recorded: mouth/tongue-moving burst, respiratory burst and irregular activity on the basis of their waveform, regularity in occurrence and concomitant muscle activity. In the GAD67-deficient mice, the occurrence of mouth/tongue-moving burst and irregular activity was inhibited to about 15 and 40% of those in the wild-type mice, respectively. Respiratory burst was slightly inhibited but the difference was not significant. Picrotoxin greatly reduced the frequency of mouth/tongue-moving burst. These results indicate that GABA is involved in rhythm generation in movement of the head region and support the hypothesis that cleft palate in the GAD67-deficient mouse is due to the impairment of mouth or tongue movement that assists palate formation.

In vitro visualization of respiratory neuron activity in the newborn mouse ventral medulla.

To clarify the neuronal organization of the respiratory center of the mouse, we analyzed the spatio-temporal pattern of respiratory neuron activity in the ventral medulla of a newborn mouse preparation, using optical recordings. We also demonstrated optical images of the respiratory activity of two different lines of knock-out mice (Tlx3-/-, Pbx3-/-) that exhibit respiratory failure leading to neonatal death from dysfunction of central respiratory neuron activity. In the wild type mice, the respiratory neuron activity in the para-facial region of the rostral medulla appeared prior to inspiratory activity in the more caudal ventrolateral medulla. This rostral to caudal activity pattern was basically preserved in Tlx3-/- mice though the activity was more dispersed and weaker than in the wild type mice. Such an activity pattern was not clearly detected in Pbx3-/- mouse preparations. The difference in the spatio-temporal pattern between Tlx3-/- and Pbx3-/- suggests different levels of functional disorder of the respiratory center.