The effect of fentanyl and morphine on neurons in the dorsal raphe nucleus in the rat: an in vitro study.

The dorsal raphe (DR) nucleus is a system of nuclei lying in the midline of the lower brainstem constituting the largest collection of serotonin-containing neurons in the brain. The DR nucleus, which is under the influence of various synaptic inputs containing the excitatory amino acids (EAAs), serotonin and noradrenaline (NA), has been reported to be involved in the process of nociception. We studied the effects of fentanyl and morphine on the firing activity of neurons within the DR nucleus in the rat brain slice preparation in vitro using extracellular recording techniques. The decreased activity of DR neurons induced by these opioids was reversed upon application of naloxone. Since almost all neurons were silent, N-methyl-D-aspartate (NMDA) and noradrenaline were used to excite the DR neurons. Serotonin contained in the DR nucleus acts as a neuromodulator to enhance the excitatory effects of NMDA. All DR neurons were excited by NMDA, whereas 72% of the neurons were excited by NA. NA has a long-lasting effect on the firing activity of the neurons, although the firing pattern was less regular than that induced by NMDA. Of the total number of the DR neurons excited by NMDA and/or NA, 71% were inhibited by fentanyl and 73% by morphine. The results support our hypothesis that these opioids inhibit the firing activity of DR neurons and, in turn, might alter nociception directly and/or indirectly by modifying the central serotonergic system.

Activity changes in rat raphe magnus neurons at different concentrations of fentanyl in vitro.

The nucleus raphe magnus (NRM) is an important descending pain inhibitory system. We postulated that the analgesic action of supraspinally administered opiates results from increased descending inhibitory control of the NRM. We tested whether fentanyl activates NRM neurons in the rat slice preparation using extra-cellular recording. Eighty-seven percent of NRM neurons (total number = 68) tested were spontaneously active with firing frequencies of 0.2-4 spikes/s in artificial cerebrospinal fluid. Application of fentanyl (0.25, 0.5, and 1 mumol/L) increased firing frequencies in 12 of 59 (20%) spontaneously active neurons. In 6 of 9 (67%) silent neurons, fentanyl induced firing activities. Naloxone (1-2 mumol/L) antagonized the increased or induced activities by fentanyl in three neurons. In 13 of 59 (22%) spontaneously active neurons, fentanyl decreased the firing frequencies. Although fentanyl was associated with increased activity in a total of 18 NRM neurons, fentanyl at a higher concentration significantly increased the number of inhibited neurons. The results indicate that fentanyl partly activates the descending inhibitory system originating from the NRM; however, at higher concentrations, it appears also to inhibit this same system.

Characterization of the mGluR(1)-mediated electrical and calcium signaling in Purkinje cells of mouse cerebellar slices.

The metabotropic glutamate receptor 1 (mGluR(1)) plays a fundamental role in postnatal development and plasticity of ionotropic glutamate receptor-mediated synaptic excitation of cerebellar Purkinje cells. Synaptic activation of mGluR(1) by brief tetanic stimulation of parallel fibers evokes a slow excitatory postsynaptic current and an elevation of intracellular calcium concentration ([Ca2+](i)) in Purkinje cells. The mechanism underlying these responses has not been identified yet. Here we investigated the responses to synaptic and direct activation of mGluR(1) using whole cell patch-clamp recordings in combination with microfluorometric measurements of [Ca2+](i) in mouse Purkinje cells. Following pharmacological block of ionotropic glutamate receptors, two to six stimuli applied to parallel fibers at 100 Hz evoked a slow inward current that was associated with an elevation of [Ca2+](i). Both the inward current and the rise in [Ca2+](i) increased in size with increasing number of pulses albeit with no clear difference between the minimal number of pulses required to evoke these responses. Application of the mGluR(1) agonist (S)-3,5-dihydroxyphenylglycine (3,5-DHPG) by means of short-lasting (5-100 ms) pressure pulses delivered through an agonist-containing pipette positioned over the Purkinje cell dendrite, evoked responses resembling the synaptically induced inward current and elevation of [Ca2+](i). No increase in [Ca2+](i) was observed with inward currents of comparable amplitudes induced by the ionotropic glutamate receptor agonist AMPA. The 3,5-DHPG-induced inward current but not the associated increase in [Ca2+](i) was depressed when extracellular Na+ was replaced by choline, but, surprisingly, both responses were also depressed when bathing the tissue in a low calcium (0.125 mM) or calcium-free/EGTA solution. Thapsigargin (10 microM) and cyclopiazonic acid (30 microM), inhibitors of sarco-endoplasmic reticulum Ca2+-ATPase, had little effect on either the inward current or the elevation in [Ca2+](i) induced by 3,5-DHPG. Furthermore, the inward current induced by 3,5-DHPG was neither blocked by 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy] ethyl-1H-imidazole, an inhibitor of store operated calcium influx, nor by nimodipine or omega-agatoxin, blockers of voltage-gated calcium channels. These electrophysiological and Ca2+-imaging experiments suggest that the mGluR(1)-mediated inward current, although mainly carried by Na+, involves influx of Ca2+ from the extracellular space.

The effect of clonidine on the activity of neurons in the rat dorsal raphe nucleus in vitro.

The dorsal raphe (DR) nucleus is a system of nuclei in the midline of the lower brainstem, which is considered one of the most important nuclei in the modulation of pain in the central nervous system. Central noradrenergic systems play an important role in the control of cardiovascular regulation and pain transmission. Clonidine, an alpha 2-adrenergic agonist is used extensively in anesthesia research. In this study, we evaluated the involvement of clonidine in the activity of DR nucleus and its possible role in pain modulation. Seventy-four neurons within the DR nucleus in the rat brainstem slice preparation were tested using extracellular recording techniques. Application of noradrenaline (NA), 50 mumol/L, induced firing activity in 68 neurons tested (92%). NA produced a regular long-lasting firing activity on the DR neurons. Fifty-six neurons (88%) previously excited by NA were inhibited by clonidine, 20 mumol/L. Clonidine suppressed the firing activity of neurons. The results indicate that the firing of DR neurons was under noradrenergic influence and was inhibited by clonidine, which in turn alters nociception by modifying the central serotonergic system.

Mechanism of cellular swelling induced by extracellular lactic acidosis in neuroblastoma-glioma hybrid (NG108-15) cells.

The mechanism of cellular swelling induced by extra-cellular lactic acidosis and the effect of diuretics were studied using neuroblastoma-glioma hybrid (NG108-15) cells. The cells were incubated in one of three lactate concentrations (0, 15, or 30 mM), each of which was randomized to one of three pH groups (7.4, 6.2, or 5.0). Analysis of the swelling was measured using a Coulter counter technique. Cellular swelling was most prominent at pH 6.2 at all lactate levels. Cellular swelling was noted to be pH dependent but not lactate dependent. The addition of 1 mM amiloride completely blocked cellular swelling, suggesting that the main mechanism of neuronal cellular swelling induced by extracellular lactic acidosis was the activation of Na+/H+ exchange. Second, three dissimilar diuretic drugs were used for cellular swelling: amiloride (Na+/H+ exchange inhibitor), mannitol (osmotic diuretic), and bumetanide (loop diuretic). Amiloride and mannitol were found effective in reducing the lactic acidosis-induced cellular swelling. Furthermore, the combination of these drugs had additive effects. However, bumetanide was not effective. The results indicate that the direct inhibition of Na+/H+ exchange and/or removal of water from the cell by mannitol was effective against cellular swelling induced by the activation of Na+/H+ exchange in NG108-15 cells.

Elevation of intradendritic sodium concentration mediated by synaptic activation of metabotropic glutamate receptors in cerebellar Purkinje cells.

Cerebellar Purkinje cells express both ionotropic glutamate receptors and metabotropic glutamate receptors. Brief tetanic stimulation of parallel fibers in rat and mouse cerebellar slices evokes a slow excitatory postsynaptic current in Purkinje cells that is mediated by the mGluR1 subtype of metabotropic glutamate receptors. The effector system underlying this mGluR1 EPSC has not yet been identified. In the present study, we recorded the mGluR1 EPSC using the whole-cell patch-clamp technique in combination with microfluorometric recordings of the intracellular sodium concentration ([Na+]i) by means of the fluorescent sodium indicator SBFI. The mGluR1 EPSC was induced by local parallel fibre stimulation in the presence of the ionotropic glutamate receptor antagonists NBQX and D-APV and the GABAA receptor antagonists bicuculline or picrotoxin. The mGluR1 EPSC was associated with an increase in [Na+]i that was restricted to a specific portion of the dendritic tree. The mGluR1 EPSC as well as the increase in [Na+]i were inhibited by the mGluR antagonist S-MCPG. In the presence of NBQX, D-APV, pictrotoxin and TTX, bath application of the selective mGluR agonist 3,5-DHPG induced an elevation in [Na+]i which extended over the whole dendritic field of the Purkinje cell. This finding demonstrates that the mGluR1-mediated postsynaptic current leads to a significant influx of sodium into the dendritic cytoplasm of Purkinje cells and thereby provides a novel intracellular signalling mechanism that might be involved in mGluR1-dependent synaptic plasticity at this synapse.

Hydrogen peroxide induces midzonal heat shock protein 72 and apoptosis in sinusoidal endothelial cells of hypoxic rat liver.

OBJECTIVE: To investigate the heat shock protein (HSP) 72 expression and apoptosis induced by hydrogen peroxide in hypoxic rat liver. DESIGN: Prospective control study using the isolated rat liver. SETTING: Animal research facility. SUBJECTS: Fasted, pathogen-free specific, male Sprague-Dawley rats. INTERVENTIONS: A low-flow hypoxia model was made by reducing an afferent pressure from 10 to 2.5 cm H2O, and by perfusing the isolated rat liver for 2 hrs. MEASUREMENT AND MAIN RESULTS: We investigated the hydrogen peroxide production by using the 2'-7' dichlorofluorescein image, the induction of HSP 72 by using immunohistochemistry, and apoptosis by using terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling method in the low flow hypoxic rat liver. In low-flow hypoxia, hydrogen peroxide production, HSP 72 expression, and apoptosis were induced in the midzone of rat liver. Prevalence of HSP 72 expression was higher in the sinusoidal endothelial cells (SEC) than in the hepatocytes. All apoptotic cells were SEC with expression of HSP 72. Hydrogen peroxide was derived from hepatocytes. Pretreatment with the specific xanthine oxidase inhibitor, sodium(-)-8-(3-methoxy-phenylsulfinylphenyl) pyrazolo [1,5-a]-1,3,5-triazine-4-olate monohydrate significantly attenuated hydrogen peroxide production, HSP 72 expression, and apoptosis of SEC in the midzone. CONCLUSION: Xanthine oxidase-dependent hydrogen peroxide induces midzonal and SEC-dominant HSP 72 expression and apoptosis in hypoxic rat liver.