Overexpression of Bcl-xl protects septal neurons from prolonged hypoglycemia and from acute ischemia-like stress.

Overexpression of Bcl-xl, a member of the Bcl-2 protein family, is reported to protect from a variety of stresses involving delayed cell death. We tested the ability of Bcl-xl overexpression to protect primary cultures of embryonic rat septal neurons subjected to one of four different stresses: 6 h of combined oxygen-glucose deprivation, which produces rapid cell death, or a 24 h exposure to hypoglycemia, hyperglycemia, or 1mM 3-nitropropionic acid (an inhibitor of mitochondrial respiration), which results in a more slowly-developing death. Prior to the stress neurons were transiently transfected to overexpress either green fluorescent protein only or green fluorescent protein along with wild-type Bcl-xl. Immediately after oxygen-glucose deprivation, many neurons expressing green fluorescent protein only showed process blebbing and disintegration, with only 49% of the initial cells remaining intact with processes. Neurons expressing both green fluorescent protein and Bcl-xl showed less damage (68% intact post-stress, P<0.05). This result indicates that Bcl-xl's saving effects are not due solely to blocking delayed (apoptotic) death, because death following oxygen-glucose deprivation was rapid and was not accompanied by increased activation of caspase-3. Bcl-xl expression also significantly protected against the hypoglycemic stress (23% intact 24 h post-stress with green fluorescent protein only, compared with 70% with Bcl-xl and green fluorescent protein), but did not protect from hyperglycemia or 3-nitropropionic acid. Thus Bcl-xl does not protect against all forms of delayed death. Bcl-xl's protective effects may include blocking early damaging events, perhaps by increasing mitochondrial function in the face of low levels of energy substrates. Bcl-xl's protective effects may require an intact electron transport chain.

Bone morphogenetic proteins (BMP6 and BMP7) enhance the protective effect of neurotrophins on cultured septal cholinergic neurons during hypoglycemia.

The effects of two bone morphogenetic proteins (BMP6, BMP7), alone and in combination with neurotrophins, were tested on cultures of embryonic day 15 rat septum. A week-long exposure to BMP6 or BMP7 in the optimal concentration range of 2-5 n M increased the activity of choline acetyltransferase (ChAT) by 1.6-2-fold, in both septal and combined septal-hippocampal cultures. The increase in ChAT activity reached significance after 4 days and continued to increase over an 11-day exposure. Under control culture conditions neither BMP significantly altered the number of cholinergic neurons, and BMP effects on ChAT activity were less than linearly additive with those of nerve growth factor. The effects of BMPs and BMP + neurotrophin combinations were also assayed under two stress conditions: low-density culture and hypoglycemia. In low-density cultures BMPs and BMP + neurotrophin combinations preserved ChAT activity more effectively than neurotrophins alone. During 24 h hypoglycemic stress, BMPs alone did not preserve ChAT activity, but BMP + neurotrophin combinations preserved ChAT activity much more effectively than neurotrophins alone. These results demonstrate that BMP6 and BMP7 enhance ChAT activity under control and low-density stress conditions, and that during a hypoglycemic stress their trophic effect requires and complements that exerted by neurotrophins.

Brief exposure to neurotrophins produces a calcium-dependent increase in choline acetyltransferase activity in cultured rat septal neurons.

We demonstrate that brief (30-min) exposure of cultured embryonic rat septal neurons to neurotrophins (NTs) increases choline acetyltransferase (ChAT) activity by 20-50% for all tested NTs (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4, each at 100 ng/ml). The increase in ChAT activity was first detected 12 h after NT exposure, persisted at least 48 h, and was not mediated by increased neuronal survival or action-potential activity. Under some conditions, the response to brief NT exposure was as great as that produced by continuous exposure. NT stimulation of ChAT activity was inhibited by inhibitors of p75- and Trk kinase-mediated signaling, by removal of extracellular Ca2+ during the period of NT exposure, and by buffering intracellular Ca2+ with BAPTA. Application of nerve growth factor and brain-derived neurotrophic factor transiently increased [Ca2+] within a subpopulation of neurons. NT stimulation of ChAT activity was not affected significantly by cyclic AMP agonists or antagonists. These findings suggest that brief exposure to NTs can have a long-lasting effect on cholinergic transmission, and that this effect requires Ca2+, but not cyclic AMP.

Resealing of transected myelinated mammalian axons in vivo: evidence for involvement of calpain.

The mechanisms underlying resealing of transected myelinated rat dorsal root axons were investigated in vivo using an assay based on exclusion of a hydrophilic dye (Lucifer Yellow-biocytin conjugate). Smaller caliber axons (<5 microm outer diameter) resealed faster than larger axons. Resealing was Ca2+ dependent, requiring micromolar levels of extracellular [Ca2+] to proceed, and further accelerated in 1 mM Ca2+. Two hours after transection, 84% of axons had resealed in saline containing 2 mM Ca2+, 28% had resealed in saline containing no added Ca2+ and only 3% had resealed in the Ca2+ buffer BAPTA (3 mM). The enhancing effect of Ca2+ could be overcome by both non-specific cysteine protease inhibitors (e.g., leupeptin) and inhibitors specific for the calpain family of Ca2+ -activated proteases. Resealing in 2 mM Ca2+ was not inhibited by an inhibitor of phospholipase A2. Resealing in low [Ca2+] was not enhanced by agents which disrupt microtubules, but was enhanced by dimethylsulfoxide (0.5-5%). These results suggest that activation of endogenous calpain-like proteases by elevated intra-axonal [Ca2+] contributes importantly to membrane resealing in transected myelinated mammalian axons in vivo.

Purification from bovine serum of a survival-promoting factor for cultured central neurons and its identification as selenoprotein-P.

We purified from bovine serum a glycoprotein that promotes the survival of rat embryonic neurons cultured from septum and other brain regions. A 40,000-fold purification was achieved by using a combination of ammonium sulfate precipitation, Zn2+ affinity chromatography, Cibacron blue 3-GA dye affinity chromatography, ABx ion exchange chromatography, and preparative PAGE. The active protein had an apparent molecular weight of 50-60 kDa. The concentration required for half-maximal survival (EC50) was 12 ng/ml ( approximately 200 pM) for the final fraction. Amino acid sequencing after cyanogen bromide cleavage yielded two sequences that are homologous to regions of deduced sequence of the selenoprotein-P (SPP) family in bovine, rat, and human. Antibodies against a synthetic peptide within the bovine SPP sequence immunoprecipitated and inhibited the survival-promoting activity of a partially purified serum fraction. The purified protein supported neuronal survival more effectively than inorganic selenium. These results suggest that SPP or an SPP-like protein contributes to the neuronal survival-promoting activity of serum.

Evidence that mitochondria buffer physiological Ca2+ loads in lizard motor nerve terminals.

1. Changes in cytosolic and mitochondrial [Ca2+] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca2+] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG-5N). Mitochondrial [Ca2+] was measured using rhod-2, bath loaded as dihydrorhod-2. 2. In response to 100-250 stimuli at 25-100 Hz the average cytosolic [Ca2+] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca2+] showed no detectable increase during the first fifteen to twenty stimuli, but after this initial delay also showed an initially rapid rise followed by a slower rate of increase. The onset of the increase in mitochondrial [Ca2+] coincided with the slowing of the rate of rise of cytosolic [Ca2+]. The peak levels of cytosolic and mitochondrial [Ca2+] both increased with increasing frequencies of stimulation. 3. When stimulation terminated, the initial rate of decay of cytosolic [Ca2+] was much more rapid than that of mitochondrial [Ca2+]. 4. After addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1-2 microM) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca2+] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca2+]. 5. These results suggest that mitochondrial uptake of Ca2+ contributes importantly to buffering presynaptic cytosolic [Ca2+] during normal neuromuscular transmission.

Stimulation-induced changes in [Ca2+] in lizard motor nerve terminals.

1. Motor axons were injected ionophoretically with one of five Ca(2+)-sensitive dyes (fluo-3, Calcium Green-2, Calcium Green-5N, fluo-3FF and Oregon Green BAPTA-5N). Changes in fluorescence (delta F/Frest) within motor terminal boutons following a single action potential and brief stimulus trains were monitored with high temporal resolution using a confocal microscope. 2. Stimulation-induced increases in delta F/Frest were confined primarily to boutons, with roughly uniform increases in all the boutons of a terminal. The increase in delta F/Frest began prior to, and decayed more slowly than, the endplate potential (EPP) recorded in the underlying muscle fibre. delta F/Frest was graded with bath [Ca2+]. Both delta F/Frest and the EPP were reduced, but not eliminated, by omega-conotoxin GVIA (5-10 microM). 3. For dyes with lower affinity for Ca2+ (e.g. Oregon Green BAPTA-5N, Kd approximately 60 microM) stimulation-induced increases in delta F/Frest were measured in the presence of the K+ channel blocker 3,4-diaminopyridine (3,4-DAP, 100 microM). During brief stimulus trains (4 at 50 Hz) in 3,4-DAP, the EPP exhibited profound depression, but the fluorescence increase associated with each stimulus showed little decrement, suggesting that depression was not mediated by a reduction in Ca2+ entry. 4. For dyes with a higher affinity for Ca2+ (e.g. fluo-3, Kd approximately 0.5-1 microM) stimulation-induced increases in delta F/Frest could also be measured in normal physiological saline. Increases in delta F/Frest were much greater with 3,4-DAP present, but the amplitude decreased with successive stimuli due to partial dye saturation. 5. Calculations suggested that following a single action potential the average [Ca2+] within a bouton increased by up to 150 nM in normal saline and 940 nM in 3,4-DAP. With low affinity dyes the delta F/Frest measured near the membrane had a higher peak amplitude and a faster early decay than that measured in the centre of the bouton, suggesting that substantial spatial [Ca2+] gradients exist within boutons for at least 15 ms following stimulation.

Spatiotemporal gradients of intra-axonal [Na+] after transection and resealing in lizard peripheral myelinated axons.

1. Post-transection changes in intracellular Na+ ([Na+]i) were measured in lizard peripheral axons ionophoretically injected with the Na(+)-sensitive ratiometric dye, sodium-binding benzofuran isophthalate (SBFI). 2. Following axonal transection in physiological saline [Na+]i increased to more than 100 mM in a region that quickly extended hundreds of micrometers from the transection site. This post-transection increase in [Na+]i was similar when the bath contained 5 microM tetrodotoxin, but was absent in Na(+)-free solution. Depolarization of uncut axons in 50 mM K+ produced little or no elevation of [Na+]i until veratridine was added. These results suggest that the post-transection increase in [Na+]i was due mainly to Na+ entry via the cut end, rather than via depolarization-activated Na+ channels. 3. The spatiotemporal profile of the post-transection increase in [Na+]i could be accounted for by movement of Na+ from the cut end with an apparent diffusion coefficient of 1.3 x 10(-5) cm2 s-1. 4. [Na+]i began to decline toward resting levels by 20 +/- 15 min (mean +/- S.D.) post-transection, except in regions of the axon within 160 +/- 85 microns of the transection site, where [Na+]i remained high. The boundary between axonal regions in which [Na+]i did or did not recover probably defines a locus of resealing of the axonal membrane. 5. [Na+]i returned to resting values within about 1 h after resealing, even in axonal regions where the normal transmembrane [Na+] gradient had completely dissipated. The recovery of [Na+]i was faster and reached lower levels than expected by diffusional redistribution of Na+ along the axon. Partial recovery occurred even in an isolated internode, indicating that the internodal axolemma can actively extrude Na+.

Neurotrophin effects on survival and expression of cholinergic properties in cultured rat septal neurons under normal and stress conditions.

These studies tested the hypothesis that survival-promoting effects of neurotrophins on basal forebrain cholinergic neurons are enhanced under stress. Septal neurons from embryonic day 14-15 rats exposed for 10-14 d to neurotrophin [nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), or neurotrophin-4 (NT-4), each at 100 ng/ml] showed a two- to threefold increase in choline acetyltransferase (ChAT) activity, with little evidence of synergistic interactions. Neurotrophins produced no significant increase in the survival of total or acetylcholinesterase (AChE)-positive neurons at moderate plating density (1200-1600 cells/mm2). However, with very low plating densities (2-28 cells/mm2) BDNF, NT-3, and NT-4 (but not NGF) increased total neuronal survival, and BDNF increased survival of AChE-positive neurons. NGF and BDNF enhanced ChAT activity and survival of cholinergic neurons after a 24 hr hypoglycemic stress, even when added 1 hr after stress onset. All four tested neurotrophins increased total neuronal survival after hypoglycemic stress. These results suggest that neurotrophins are important for preservation of central cholinergic function under stress conditions, with different neurotrophins protecting against different stresses. The stress-associated survival-promoting effects of neurotrophins were not limited to the cholinergic subpopulation.

Electrical and morphological factors influencing the depolarizing after-potential in rat and lizard myelinated axons.

1. Intra-axonal recording and electron microscopy were applied to intramuscular myelinated axons in lizards and rats to investigate factors that influence the amplitude and time course of the depolarizing after-potential. 2. Depolarizing after-potentials in lizard axons had larger peak amplitudes and longer half-decay times than those recorded in rat axons (mean values 10 mV, 35 ms in lizard; 3 mV, 11 ms in rat). These differences were not due to differences in temperature, resting potential or action potential amplitude or duration. 3. For a given axon diameter, the myelin sheath in lizard fibres was thinner and had fewer wraps than in rat fibres. There was no significant difference in myelin periodicity. Calculations suggest that the thinner myelin sheath accounts for < 30% of the difference between depolarizing after-potential amplitudes recorded in lizard and rat axons. 4. Consistent with a passive charging model for the depolarizing after-potential, the half-time of the passive voltage transient following intra-axonal injection of current was shorter in rat than in lizard axons. 5. Aminopyridines prolonged the falling phase of the action potential and increased the amplitude of the depolarizing after-potential in both types of axon. 6. During repetitive stimulation the depolarizing after-potentials following successive action potentials exhibited little or no summation. Axonal input conductance in the interspike interval increased during the train. 7. These findings suggest that the amplitude and time course of the depolarizing after-potential are influenced not only by the passive properties of the axon and myelin sheath, but also by persisting activation of axolemmal K+ channels following action potentials.

Changes in the response of cultured septal cholinergic neurons to nerve growth factor exposure and deprivation during the first postnatal month.

The effects of nerve growth factor (NGF) and a blocking anti-NGF antibody were studied in cultures plated from postnatal day 1-28 (P1-P28) rat septum and maintained 3 weeks in vitro. 7S NGF (100 ng/ml = 0.75 nM) increased choline acetyltransferase (ChAT) activity in P7-P21 cultures. The largest increase was measured in P7-P14 cultures, where NGF addition produced ChAT activities 5-12 times higher than those measured in cultures grown in anti-NGF antibody. NGF also increased the number of acetylcholinesterase (AChE)-positive neurons in P7-P14 cultures. To determine whether this increase was due to enhanced survival of cholinergic neurons or simply to enhanced AChE expression, we examined cultures to which NGF was added only after an initial 1-2-week exposure to anti-NGF antibody. This delayed addition of NGF also increased ChAT activity and the number of AChE-positive neurons, indicating that cholinergic neurons survived the initial exposure to anti-NGF antibody. Thus even during a period when ChAT activity was most sensitive to NGF, postnatal septal cholinergic neurons did not require NGF for survival in vitro.

Activation of internodal potassium conductance in rat myelinated axons.

1. Voltage changes associated with currents crossing the internodal axolemma were monitored using a microelectrode inserted into the myelin sheath (peri-internodal region) of rat phrenic nerve fibres. This microelectrode was also used to change the potential and the ionic environment in the peri-internodal region. 2. Following stimulation of the proximal nerve trunk, the peri-internodal electrode recorded a positive-going action potential whose amplitude increased (up to 75 mV) with increasing depth of microelectrode penetration into the myelin. The resting potential recorded by the peri-internodal electrode remained within 4 mV of bath ground. 3. Confocal imaging of fibres injected peri-internodally with the fluorescent dye Lucifer Yellow revealed a staining pattern consistent with spread of dye throughout the myelin sheath of the injected internode. 4. After ionophoresis of K+ (but not Na+) into the peri-internodal region, the action potential was followed by a prolonged negative potential (PNP) lasting hundreds of milliseconds to several seconds. The duration of the PNP increased as the frequency of stimulation decreased. PNPs could also be evoked by sub-threshold depolarization of the internodal axolemma with peri-internodally applied current pulses. In the absence of action potentials or applied depolarization PNPs sometimes appeared spontaneously. 5. Peri-internodal application of Rb+ also produced evoked and spontaneous PNPs. These PNPs had longer durations (up to 20 s) than those recorded from K(+)-loaded internodes. 6. Spontaneous action potentials sometimes appeared during the onset of the PNP, suggesting that PNPs are associated with depolarization of the underlying axon. 7. Passage of current pulses during the PNP demonstrated that the PNP is associated with an increased conductance of the pathway linking the peri-internodal recording site to the bath. At least part of this conductance increase occurs across the internodal axolemma, since peri-internodally recorded action potentials evoked during the PNP had larger amplitudes than those evoked before or after the PNP. 8. PNPs were suppressed by tetraethylammonium (TEA, 10-20 mM) and by 4-aminopyridine (1 mM). 9. These results suggest that the PNPs recorded in K(+)- or Rb(+)-loaded myelin sheaths are produced by a regenerative K+ or Rb+ current that enters the internodal axolemma via K+ channels opened by action potentials or subthreshold depolarizations. 10. When normal extracellular [K+] was preserved (by using Na+ rather than K+ salts in the peri-internodal electrode), action potentials recorded within the myelin sheath were instead followed by a brief, positive after-potential that was inhibited by TEA.(ABSTRACT TRUNCATED AT 400 WORDS)

Posttetanic hyperpolarization produced by electrogenic Na(+)-K+ pump in lizard axons impaled near their motor terminals.

1. The hyperpolarization that follows tetanic stimulation was recorded intra-axonally from the internodal region of intramuscular myelinated motor axons. 2. The peak amplitude of the posttetanic hyperpolarization (PTH) that followed stimulation at 20-100 Hz for < or = 35 s increased with increasing train duration, reaching a maximum of 22 mV. PTH decayed over a time course that increased from tens to hundreds of seconds with increasing train duration. For a given frequency of stimulation the time integral of PTH was proportional to the number of stimuli in the train, averaging 3-4 mV.s per action potential. 3. Ouabain (0.1-1 mM) and cyanide (1 mM) depolarized the resting potential and abolished PTH. Tetanic stimulation in ouabain was followed by a slowly decaying depolarization (probably due to extra-axonal K+ accumulation) whose magnitude and duration increased as the duration of the train increased. 4. Axonal input resistance showed no consistent change during PTH in normal solution but increased during PTH in the presence of 3 mM Cs+ (which blocks axonal inward rectifier currents). 5. PTH was abolished when bath Na+ was replaced by Li+ or choline. PTH persisted after removal of bath Ca2+ and addition of 2 mM Mn2+. 6. Removal of bath K+ abolished the PTH recorded after brief stimulus trains and greatly reduced the duration of PTH recorded after longer stimulus trains. 7. A brief application of 10 mM K+, which normally depolarizes axons, produced a ouabain-sensitive hyperpolarization in axons bathed in K(+)-free solution. 8. These observations suggest that in these myelinated axons PTH is produced mainly by activation of an electrogenic Na(+)-K(+)-ATPase, rather than by changes in K+ permeability or transmembrane [K+] gradients. This conclusion is supported by calculations showing agreement between estimates of Na+ efflux/impulse based on PTH measurements and estimates of Na+ influx/impulse based on nodal voltage-clamp measurements. Pump activity also appears to contribute to the resting potential. 9. The stimulus intensity required to initiate a propagating action potential increased during PTH but decreased during the posttetanic depolarization recorded in ouabain. Thus changes in axonal excitability after tetanic stimulation correlate with changes in the posttetanic membrane potential. 10. Action potentials that propagated during PTH had a larger peak amplitude and were followed by a larger and longer depolarizing afterpotential than action potentials elicited at the resting potential. This enhancement of the depolarizing afterpotential is consistent with previous reports of an increased superexcitable period after action potentials evoked during PTH.

Reversibility of nerve growth factor's enhancement of choline acetyltransferase activity in cultured embryonic rat septum.

Choline acetyltransferase (ChAT) activity and survival of acetylcholinesterase (AChE)-positive neurons were measured in low-density cultures of embryonic (Day 14-15) rat septum exposed to various sequences of nerve growth factor (NGF) exposure and deprivation for up to 7 weeks in vitro. Most septal cultures grown 4-5 weeks with no exogenous NGF (including exposure to monoclonal or polyclonal antibodies against NGF) retained both a basal ChAT activity and the ability to increase ChAT activity in response to subsequently added NGF. When cultures were exposed to NGF (7S, 0.75 nM) for 2-3 weeks and then deprived of NGF for 2 weeks, ChAT activity fell gradually, but the number of AChE-positive neurons remained unchanged, and in many cases ChAT activity could be restored by subsequent re-exposure to NGF. Thus NGF's enhancement of ChAT activity in embryonic septal neurons in vitro is largely reversible and is not mediated by differential survival of cholinergic neurons.

Neurotoxic components in normal serum.

Serum neurotoxicity was studied by adding whole or fractionated serum (adult human, adult horse, or newborn calf) to neuron-rich cultures prepared from various regions of embryonic (Days 14-15) rat brain, including spinal cord, ventral mesencephalon, cerebellum, septum, and striatum. Effects of serum were also tested on several types of embryonic non-neuronal cells (skeletal muscle myotubes, cardiac muscle myocytes, and fibroblasts from skin and lung). Serum concentrations of 50% or more killed more than 95% of all neurons within 3 days. Serum concentrations as low as 10% also killed some neurons, especially those from cerebellum. Septal, cerebellar, and spinal cord neurons were more sensitive than striatal or mesencephalic neurons. All the tested non-neuronal cells survived much better than neurons at serum concentrations of 20% or more. Neurotoxicity was present in both fresh (human) and previously frozen (human and animal) sera, and affected both young (4 days in vitro) and older (42 days in vitro) cultures. Neurotoxicity was greatly diminished by heating the serum to 56 degrees C for 30 min. Experiments indicated that serum toxicity was not due to lipoprotein, complement, or tumor necrosis factor. All serum neurotoxicity was retained by an ultrafilter with a nominal molecular weight cutoff of 10 kDa. The profile of neurotoxicity following gel filtration at neutral pH was variable, with high toxicity most consistently observed in fractions with apparent molecular weights exceeding 100 kDa, and variable degrees of toxicity at lower molecular weights.

Rat embryonic septal neurons survive and express cholinergic properties in isolation and without nerve growth factor.

We studied survival and expression of cholinergic properties in embryonic septal neurons grown in very low density microcultures (1-7 cells per Terasaki well). Even in cultures containing only a single neuron, at least 10% of plated neurons survived for 2 weeks or more in medium containing fetal calf serum or an acid-stable fraction (55,000 Da) of horse serum. Of these surviving neurons, 30-40% stained positively for acetylcholinesterase (AChE) or nerve growth factor (NGF) receptor, even though the culture medium lacked detectable levels of NGF, brain-derived neurotrophic factor, and fibroblast growth factor. Addition of NGF or an antibody against NGF had no effect on either neuronal survival or the percentage of neurons staining positively for AChE or NGF receptor after 18-20 days in vitro. There was no cell division in medium containing the serum fraction, but when 10% fetal calf serum was present cell division occurred in some of the cultures, and in half of these cases at least one of the clonal progeny became AChE-positive. These results demonstrate that some embryonic septal cells can survive at least 2 weeks and develop cholinergic neuronal properties in the absence of other cells or NGF.

Differential effects of insulin on choline acetyltransferase and glutamic acid decarboxylase activities in neuron-rich striatal cultures.

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.

Evidence that action potentials activate an internodal potassium conductance in lizard myelinated axons.

1. We have studied action potentials and after-potentials evoked in the internodal region of visualized lizard intramuscular nerve fibres by stimulation of the proximal nerve trunk. Voltage recordings were obtained using microelectrodes inserted into the axon (intra-axonal) or into the layers of myelin (peri-internodal), with the goal of studying conditions required to activate internodal K+ currents. 2. Peri-internodal recordings made using K2SO4-, KCl- or NaCl-filled electrodes exhibited a negligible resting potential (less than 2 mV), but showed action potentials with peak amplitudes of up to 78 mV and a duration less than or equal to that of the intra-axonally recorded action potential. 3. Following ionophoretic application of potassium from a peri-internodal microelectrode, the peri-internodal action potential was followed by a prolonged (hundreds of milliseconds) negative plateau. This plateau was not seen following peri-internodal ionophoresis of sodium. The prolonged negative potential (PNP) was confined to the K(+)-injected internode: it could be recorded by a second peri-internodal microelectrode inserted into the same internode, but not into an adjacent internode. 4. The peri-internodally recorded PNP was accompanied by an equally prolonged intra-axonal depolarizing after-potential, and by an increase in the conductance of the internodal axolemma. However, the K+ ionophoresis that produced the PNP had little or no detectable effect on the intra-axonally or peri-internodally recorded resting potential or action potential. These findings suggest that the PNP is generated by an inward current across the axolemma of the K(+)-injected internode, through channels opened following the action potential. 5. Following peri-internodal K+ ionophoresis a PNP could also be evoked by passage of depolarizing current pulses through an intra-axonal electrode or by passage of negative current pulses through an electrode in the K(+)-filled peri-internodal region. The threshold for evoking a PNP was less than the threshold for evoking an action potential, and the PNP persisted in 10 microM-tetrodotoxin. Thus the PNP is evoked by depolarization of the axolemma rather than by Na+ influx. 6. The PNP was reversibly blocked by tetraethylammonium (TEA, 2-10 mM), but was not blocked by 100 microM-3,4-diaminopyridine or 5 mM-4-aminopyridine.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane resealing in cultured rat septal neurons after neurite transection: evidence for enhancement by Ca(2+)-triggered protease activity and cytoskeletal disassembly.

Neurites of cultured septal neurons were transected with a laser under sterile conditions, and the subsequent membrane resealing was assayed using a dye exclusion method. In agreement with findings in other preparations, Ca2+ enhanced resealing: in normal culture medium the percentage of lesioned neurons that resealed within 20-30 min after transection increased with increasing bath [Ca2+] over the range 10(-7) to 2 x 10(-3) M; about 75% of cells resealed in 2 mM Ca2+. Mn2+ and Sr2+ also enhanced resealing, but Mg2+ inhibited it. The percentage of resealing neurons was sensitive to agents known to alter the stability of cytoskeletal components. Agents that tend to disassemble microtubules and/or neurofilaments (e.g., colchicine, low-ionic-strength media) strongly promoted resealing, whereas treatments that tend to stabilize microtubules (taxol, Mg2+) inhibited resealing. Addition of exogenous proteases (papain, trypsin, or dispase) enhanced resealing, whereas inhibitors of cysteine proteases (including a specific inhibitor of calpain, a Ca-activated neutral protease) strongly inhibited resealing. Calmodulin inhibitors inhibited resealing, consistent with reports that calmodulin facilitates calpain-mediated proteolysis of fodrin, a component of the cortical cytoskeleton. Based on these results, we hypothesize that one of the major mechanisms involved in resealing is activation of endogenous proteases by Ca2+ entry into the injured neurite. The resulting changes in the cellular cytoskeleton might promote fusion and resealing of the cut ends of the plasma membrane by enhancing membrane mobility and/or by removing structures that normally prevent membrane-membrane contact.

Transplantation of human fetal dopamine cells for Parkinson's disease. Results at 1 year.

In an effort to improve the clinical signs of Parkinson's disease, we have implanted mesencephalic dopamine cells from a 7-week human embryo into the caudate and putamen of a 52-year-old man with Parkinson's disease. Fetal tissue was obtained from elective abortion. The woman and the patient with Parkinson's disease were unknown to each other. The woman gave specific consent and was not paid. The patient had a 20-year history of parkinsonism treated with multiple drug therapies including levodopa/carbidopa (Sinemet) every 2 1/2 hours. His symptoms were worse on the left side. For 5 months prior to transplantation, the patient underwent clinical evaluations by both a neurologist and a computer system installed in his home for daily measurement of walking and hand movements. Preoperative positron emission tomographic scanning with 6-L[18F]fluorodopa (fluorodopa) demonstrated severe dopamine depletion bilaterally. Fetal tissue was matched to the patient for ABO blood antigens, and maternal serum was screened for hepatitis B and human immunodeficiency virus type 1 prior to surgery. Fetal tissue was implanted stereotactically throughout the caudate and putamen on the right side of the brain via 10 needle tracks. The patient was not immunosuppressed. Results 12 months after surgery showed 42% improvement in left-hand speed before the first morning dose of drug and 40% greater response to drug therapy. Right-hand speed increased 15% before drug therapy and 23% after drug therapy. Reaction time was unaffected. Walking speed increased 33% after drug administration, although walking speed before the first morning dose of drugs declined 40%. Walking speed on an all-day basis improved 17%.(ABSTRACT TRUNCATED AT 250 WORDS)