Fusao Nakamura

Assessment of microPET performance in analyzing the rat brain under different types of anesthesia: comparison between quantitative data obtained with microPET and ex vivo autoradiography

MicroPET (positron emission tomography) has been implemented for use in experiments with small animals. However, the quantification and optimal conditions for scanning are not established yet. The aim of this study was to compare the results obtained by microPET with those by ex vivo autoradiography of rat brain slices, based on the 2-[18F]fluoro-2-deoxy-D-glucose (FDG) method, and to establish the optimal conditions for scanning. As an example, we examined glucose metabolism in the rat brain under 6 types of anesthesia and in the conscious state. The scanning conditions for the rat brain were (1) use of a 4-mm-thick leaden jacket, (2) an energy window of 350-650 keV, and (3) a coincidence time window of 6 ns. Under these conditions, the quantitative ROI data from microPET showed a good correlation with the corresponding ROI data from FDG autoradiography in the animal study (r2=0.81). With our protocol, when anesthesia was started 40 min after the FDG injection, the glucose metabolism was almost the same as that in the conscious rat brain.

Establishment and assessment of a rat model of fatigue

To establish an animal model of fatigue, we kept rats in a cage filled with water to a height of 1.5 cm. We selected a weight-loaded forced swimming test for evaluation of the extent of fatigue. Animals kept in the wet cage for 5 days showed a reduction in 2-[18F]fluoro-2-deoxy-D-glucose uptake into their brain. The session for 1 day showed significantly increased 5-hydroxyindoleacetic acid (5-HIAA)/5-hydroxytryptamine (5-HT) and [3,4-dihydroxyphenyl-acetic acid (DOPAC)+homovanillic acid (HVA)]/dopamine (DA) ratios in all brain regions, but the session for 5 days showed the restoration of the 5-HIAA/5-HT ratio in the hippocampus and hypothalamus and in the (DOPAC+HVA)/DA ratio in the striatum and hypothalamus. Our data suggest that decreased glucose uptake and insufficient serotonin and dopamine turnover introduced by deprivation of rest were correlated with central fatigue.

MicroPET detection of enhanced 18F-FDG utilization by PKA inhibitor in awake rat brain

To obtain PET imaging of glucose metabolism in the brains of conscious rats, a method of rat head fixation was developed. PET measurement with microPET was performed for 60 min after 18F-FDG injection. Significant enhancement of glucose utilization in the right striatum was observed with infusion of Rp-adenosine-3,5-cyclic phosphorothioate triethylamine (Rp-cAMPS). FDG uptake increments were also seen in the ipsilateral frontal cortex and thalamus. As initial FDG uptake in the brain was not significantly altered by Rp-cAMPS, increased glucose metabolism might be due to an increase in the phosphorylation rate by hexokinase rather than the delivery process from plasma to the brain. In contrast to awake rats, the effect of Rp-cAMPS was abolished by anesthesia using chloral hydrate, indicating that neuronal activity has an important role in short term regulation of hexokinase activity through the cAMP/PKA system in the brain. These results strongly demonstrated the value of measuring glucose utilization in the brains of conscious rats.

Synergetic activation of outwardly rectifying Cl− currents by hypotonic stress and external Ca2+ in murine osteoclasts

1 An outwardly rectifying Cl− (ORCl) current of murine osteoclasts was activated by hypotonic stimulation. The current was characterized by rapid activation, little inactivation, strong outward rectification, blockage by DIDS and permeability to organic acids (pyruvate and glutamate). 2 The hypotonically activated ORCl current was inhibited by intracellular dialysis with an ATP‐free pipette solution, but not by replacement of ATP with a poorly hydrolysable ATP analogue adenosine 5′‐O‐(3‐thiotriphosphate). The current amplitude was reduced when intracellular alkalinity increased over the pH range 6.6–8.0. 3 Intracellular application of cytochalasin D occasionally activated the ORCl current without hypotonic stress, but inhibited activation of the ORCl current by hypotonic stimulation. The hypotonically activated ORCl current was unaffected by a non‐actin‐depolymerizing cytochalasin, chaetoglobosin C, but partially inhibited by deoxyribonuclease I. 4 Removal of extracellular Ca2+ inhibited activation of the ORCl current by hypotonic shock, but did not reduce the current once activated. The hypotonically activated ORCl current was partially decreased by intracellular dialysis with 20 mm EGTA. 5 With 10 mm Ca2+ in the extracellular medium, the ORCl current was activated in response to more minor decreases in osmolarity than with 1 mm Ca2+. The increased sensitivity to hypotonicity was mimicked by increasing the intracellular Ca2+ level (pCa 6.5). 6 These results suggest that hypotonic stimulation and a rise in the extracellular Ca2+ level synergistically activate the ORCl channel of murine osteoclasts, and that the activating process is modified by multiple intracellular factors (pH, ATP and actin cytoskeletal organization).

Differential effect of high extracellular Ca2+ on K+ and Cl- conductances in murine osteoclasts.

Abstract. Effects of the extracellular Ca2+ concentration ([Ca2+]o) on whole cell membrane currents were examined in mouse osteoclastic cells generated from bone marrow/stromal cell coculture. The major resting conductance in the presence of 1 mm Ca2+ was mediated by a Ba2+-sensitive, inwardly rectifying K+ (IRK) current. A rise in [Ca2+]o (5–40 mm) inhibited the IRK current and activated an 4,4′-diisothiocyano-2,2′-stilbenedisulfonate (DIDS)-sensitive, outwardly rectifying Cl− (ORCl) current. The activation of the ORCl current developed slowly and needed higher [Ca2+]o than that required to inhibit the IRK current. The inhibition of the IRK current consisted of two components, initial and subsequent late phases. The initial inhibition was not affected by intracellular application of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) or guanosine 5′-O-(2-thiodiphosphate) (GDPβS). The late inhibition, however, was enhanced by GTPγS and attenuated by GDPβS, suggesting that GTP-binding proteins mediate this inhibition. The activation of the ORCl current was suppressed by pretreatment with pertussis toxin, but not potentiated by GTPγS. An increase in intracellular Ca2+ level neither reduced the IRK current nor activated the ORCl current. Staurosporine, an inhibitor for protein kinase C, did not modulate the [Ca2+]o-induced changes in the IRK and ORCl conductances. These results suggest that high [Ca2+]o had a dual action on the membrane conductance of osteoclasts, an inhibition of an IRK conductance and an activation of an ORCl conductance. The two conductances modulated by [Ca2+]o may be involved in different phases of bone resorption because they differed in Ca2+ sensitivity, temporal patterns of changes and regulatory mechanisms.

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg2+, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 microM ACPD and in 7 of 12 cells with 0.5 microM ACPD. The EPSP amplitudes with 5 and 0.5 microM ACPD were 142 +/- 10% (mean +/- S.E.M., n = 19) and 130 +/- 13% (n = 12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 microM ACPD were not significantly different in cells perfused with the low Ca(2+)-high Mg2+ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Glutamate-induced inhibition of paired pulse facilitation of monosynaptic excitatory post-synaptic potentials in frog spinal motoneurons

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg2+. In most cells perfused with Mg(2+)-free or high Ca(2+)-Mg2+ solutions, the glutamate potential was accompanied by a reduction in peak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg(2+)-free and high Ca(2+)-Mg2+ solutions, and in most cells with high Mg2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 microM), kainate (50-100 microM), quisqualate (5-50 microM) and L-2-amino-4-phosphonobutyrate (L-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 microM) or 2-amino-5-phosphonovalerate (APV, 250 microM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreased.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-cultured development of basic electrophysiological properties of spinal neurons obtained from rat embryo.

Basic electrical profiles of cultured neurons are modified by multiple factors, such as cell growth, differentiation and cell damage from the isolation procedure. In the present study, we assessed development of electrophysiological properties of rat spinal neurons over the late embryonic and early postnatal period in a neuron-enriched culture. After recovery from acute damage within 2 days after plating, the input conductance and amplitudes of voltage-gated Na(+) and K(+) currents increased parallel to the increase in the cell capacitance. Whether this depended on the period or the growth of the cell area was estimated by normalizing the parameters with the cell capacitance. The input conductance per unit area, the membrane time constant and the Na(+) current density remained constant for two weeks. However, densities of two types of outward K(+) currents, an A-current and a delayed rectifier, required 3--5 days to reach the maximum, although neither thresholds for activation nor sensitivities to blockers (TEA and 4AP) altered. The hyperpolarizing shift of the resting membrane potential became stabilized within 6--8 days, suggesting that the development of the K(+) currents underlay the shift. These results show that passive electrical properties and voltage-gated currents of rat spinal neurons in the neuron-rich culture differ in temporal patterns of development but stabilize at the latest within a week, corresponding to the day of birth.

Enhancement of monosynaptic excitatory postsynaptic potentials by glutamate in frog spinal motoneurons

We analyzed glutamate-induced enhancement of the amplitude of monosynaptic excitatory postsynaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Low concentrations (0.1-0.3 mM) of glutamate, which produced small depolarization, often enhanced EPSP associated with inhibition of a paired pulse facilitation and increased occurrence of spontaneous EPSPs. With 1 mM glutamate, transient enhancement of EPSP was seen in some cells during the early phase or prior to large depolarization, even when input conductance was increased. Transient or sustained enhancement of EPSP was occasionally seen with N-methyl-D-aspartate, kainate and quisqualate, but not with L-2-amino-4-phosphonobutyrate. The results suggest that glutamate enhanced release of excitatory transmitters at low concentrations that apparently did not affect the postsynaptic membrane.

Diversity and development of potassium currents in rat spinal neurons

1Dept. of Physiology, 2Dept. of Neuropsychiatry & 3Biomedical Imaging Research Center, Fukui Medical School, Matsuokacho, Yoshida-gun, Fukui 910-1193 The activity of neurons was evaluated in sagittally-sectioned brain slices of the rat using the [18F]FDG ([18F]2-fluoro-2deoxy-D-glucose) produced by a cyclotron. The neuronal activity, which is proportional to the amount of [18F]FDG uptake, was monitored every 20 min up to 7 h in five representative brain regions: frontal cortex, striatum, thalamus, hippocampus and cerebellum. Glucose utilization in these regions was constant under control conditions. When brain slices were exposed to 3 ,u M TTX (tetrodotoxin), the uptake of glucose was greatly inhibited. This inhibition was reversed when TTX was washed out. Some uptake was resistant to TTX which may be ascribed to glucose metabolism in neurons and glial cells not associated with neuronal firing. Remaining functional synapses were not abundant in brain slices, because the glucose uptake showed only a slight decrease when the synaptic activity was suppressed in Ca2+-free, high-Mg2+ solution. A neurotoxic effect, irreversible decrease in the glucose uptake, was produced when 0.1-l mM Cdz+ was applied to brain slices. These findings indicate that the present method may be useful for the examination of short term neurotoxicity induced by test substances.