NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Activation of Cl− Currents in Cultured Rat Retinal Pigment Epithelial Cells by Intracellular Applications of Inositol-1,4,5-triphosphate: Differences Between Rats with Retinal Dystrophy (RCS) and Normal Rats

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl⁻-currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 μm inositol-1,4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl⁻ currents, blockable by 1 mm 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). IP3 activated Cl⁻ currents in the presence of a high concentration of the calcium chelator BAPTA (10 mm) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10⁻⁵ m Ca²⁺ via the patch pipette also led to a transient activation of Cl⁻ currents. When the cells were preincubated in a bath solution containing thapsigargin (1 μm) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca²⁺ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl⁻-current activation induced by IP3 which could be reduced by nifedipine (1 μm). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3. Received: 17 July 1995/Revised: 31 January 1996     

Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Halcyon days with Bill Arnold

The circumstances that led to the discovery that plants luminesce after they are illuminated are described, as are other discoveries that would not have been possible were it not for the fortuitous association I had with my dear and most admirable friend, W.A. Arnold, to whom this special issue is dedicated.     

Heartbeat control in the medicinal leech: A model system for understanding the origin,coordination, and modulation of rhythmic motor patterns

We have analyzed in detail the neuronal network that generates heartbeat in the leech. Reciprocally inhibitory pairs of heart interneurons form oscillators that pace the heartbeat rhythm. Other heart interneurons coordinate these oscillators. These coordinating interneurons, along with the oscillator interneurons, form an eight-cell timing oscillator network for heartbeat. Still other interneurons, along with the oscillator interneurons, inhibit heart motor neurons, sculpting their activity into rhythmic bursts. Critical switch interneurons interface between the oscillator interneurons and the other premotor interneurons to produce two alternating coordination states of the motor neurons. The periods of the oscillator interneurons are modulated by endogenous RFamide neuropeptides. We have explored the ionic currents and graded and spike-mediated synaptic transmission that promote oscillation in the oscillator interneurons and have incorporated these data into a conductance-based computer model. This model has been of considerable predictive value and has led to new insights into how reciprocally inhibitory neurons produce oscillation. We are now in a strong position to expand this model upward, to encompass the entire heartbeat network, horizontally, to elucidate the mechanisms of FMRFamide modulation, and downward, to incorporate cellular morphology. By studying the mechanisms of motor pattern formation in the leech, using modeling studies in conjunction with parallel physiological experiments, we can contribute to a deeper understanding of how rhythmic motor acts are generated, coordinated, modulated, and reconfigured at the level of networks, cells, ionic currents, and synapses. © 1995 John Wiley & Sons, Inc.     

Spontaneous calcium transients regulate neuronal plasticity in developing neurons

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10⁴ times slower than that of action potentials. © 1995 John Wiley & Sons, Inc.     

The weaver mutant mouse as a model of nigrostriatal dysfunction

The weaver mutant mouse has a genetic defect that results in the loss of dopamine neurons in the nigrostriatal pathway. Striatal tyrosine hydroxylase and dopamine content are reduced by 60–70%, and dopamine uptake is reduced by as much as 95%. Deficits in all three of these striatal dopamine markers are seen as early as postnatal d 3. The striatal dopamine systems in the weaver apparently have the ability to compensate for this dopamine deficit. Thus, in the weaver, in vitro resting release, as well as amphetamine-evoked fractional release of endogenous dopamine are increased. An additional change seen in the weaver striatum is an elevated serotonin content. These alterations may play an adaptive role in attempting to compensate for the dopamine loss. In summary, the weaver mutant mouse has dramatic deficits in the nigrostriatal pathway, but also seems to develop certain adaptive mechanisms in dopaminergic and other transmitter systems that may compensate functionally for the dopamine deficit. Thus, the weaver mouse provides a unique animal model for studying naturally induced neuronal degeneration that complements those models using surgical and pharmacological protocols.     

The role of neurotrophins in the developing nervous system

Neurotrophins were originally identified by their ability to promote the survival of developing neurons. However, recent work on these proteins indicates that they may also influence the proliferation and differentiation of neuron progenitor cells and regular several differentiated traits of neurons throughout life. Moreover, the effects of neurotrophins on survival have turned out to be more complex than originally thought. Some neurons switch their survival requirements from one set of neurotrophins to another during development, and several neurotrophins may be involved in regulating the survival of a population of neurons at any one time. Much of our understanding of the developmental physiology of neurotrophins has come from studying neurons of the peripheral nervous system. Because these neurons and their progenitors are segregated into anatomically discrete sites, it has been possible to obtain these cell for in vitro experimental studies from the earliest stage of their development. The recent generation of mice having null mutations in the neurotrophin and neurotrophin receptor genes has opened up an unparalleled opportunity to assess the physiological relevance of the wealth of data obtained from these in vitro studies. Here I provide a chronological account of the effects of members of the NGF family of neurotrophins on cells of the neural lineage with special reference to the peripheral nervous system. 1994 John Wiley & Sons, Inc.     

Second messenger and protein phosphorylation mechanisms underlying opiate addiction: Studies in the rat locus coeruleus

We have studied the role of second messenger and protein phosphorylation pathways in mediating changes in neuronal function associated with opiate addiction in the rat locus coeruleus. We have found that chronic opiates increase levels of the G-protein subunits Gi and Go, adenylate cyclase, cyclic AMP-dependent protein kinase, and a number of phosphoproteins (including tyrosine hydroxylase) in this brain region. Electrophysiological data have provided direct support for the view that this up-regulation of the cyclic AMP system contributes to opiate tolerance, dependence, and withdrawal exhibited by these neurons. As the adaptations in G-proteins and the cyclic AMP system appear to occur at least in part at the level of gene expression, current efforts are aimed at identifying the mechanisms, at the molecular level, by which opiates regulate the expression of these intracellular messenger proteins in the locus coeruleus. These studies will lead to an improved understanding of the biochemical basis of opiate addiction.Special issue dedicated to Dr. Paul Greengard     

Insulin-like growth factors: Putative muscle-derived trophic agents that promote motoneuron survival

Treatment of chick embryos in ovo with IGF-I during the period of normal, developmentally regulated neuronal death (embryonic days 5–10) resulted in a dose-dependent rescue of a significant number of lumbar motoneurons from degeneration and death. IGF-II and two variants of IGF-I with reduced affinity for IGF binding proteins, des(1-3) IGF-I and long R³ IGF-I, also elicited enhanced survival of motoneurons equal to that seen in IGF-I-treated embryos. IGF-I did not enhance mitogenic activity in motoneuronal populations when applied to embryos during the period of normal neuronal proliferation (E2-5). Treatment of embryos with IGF-I also reduced two types of injury-induced neuronal death. Following either deafferentation or axotomy, treatment of embryos with IGF-I rescued approximately 75% and 50%, respectively, of the motoneurons that die in control embryos as a result of these procedures. Consistent with the survival-promoting activity on motoneurons in ovo, IGF-I, -II, and des(1-3) IGF-I elevated choline acetyltransferase activity in embryonic rat spinal cord cultures, with des (1-3) IGF-I demonstrating 2.5 times greater potency than did IGF-I. A single addition of IGF-I at culture initiation resulted in the maintenance of 80% of the initial ChAT activity for up to 5 days, during which time ChAT activity in untreated control cultures fell to 9%. In summary, these results demonstrate clear motoneuronal trophic activity for the IGFs. These findings, together with previous reports that IGFs are synthesized in muscle and may participate in motoneuron axonal regeneration and sprouting, indicate that these growth factors may have an important role in motoneuron development, maintenance, and recovery from injury. © 1993 John Wiley & Sons, Inc.