Ronald J. Knox

Brain‐Derived Neurotrophic Factor Induces Excitotoxic Sensitivity in Cultured Embryonic Rat Spinal Motor Neurons Through Activation of the Phosphatidylinositol 3‐Kinase Pathway

Abstract: Neurotrophic factors (NTFs) can protect against or sensitize neurons to excitotoxicity. We studied the role played by various NTFs in the excitotoxic death of purified embryonic rat motor neurons. Motor neurons cultured in brain‐derived neurotrophic factor, but not neurotrophin 3, glial‐derived neurotrophic factor, or cardiotrophin 1, were sensitive to excitotoxic insult. BDNF also induces excitotoxic sensitivity (ES) in motor neurons when BDNF is combined with these other NTFs. The effect of BDNF depends on de novo protein and mRNA synthesis. Reagents that either activate or inhibit the 75‐kDa NTF receptor p75NTR do not affect BDNF‐induced ES. The low EC50 for BDNF‐induced survival and ES suggests that TrkB mediates both of these biological activities. BDNF does not alter glutamate‐evoked rises of intracellular Ca2+, suggesting BDNF acts downstream. Both wortmannin and LY294002, which specifically block the phosphatidylinositol 3‐kinase (PI3K) intracellular signaling pathway in motor neurons, inhibit BDNF‐induced ES. We confirm this finding using a herpes simplex virus (HSV) that expresses the dominant negative p85 subunit of PI3K. Infecting motor neurons with this HSV, but not a control HSV, blocks activation of the PI3K pathway and BDNF‐induced ES. Through the activation of TrkB and the PI3K signaling pathway, BDNF renders developing motor neurons susceptible to glutamate receptor‐mediated cell death.

Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro

Abstract : We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non‐NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration‐ and time‐dependent manner, which can be blocked by receptor subtype‐specific antagonists. subunit‐specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca2+ is required for excitotoxicity, and the route of entry initiated by activation of non‐NMDA, but not NMDA, receptors is L‐type Ca2+ channels. Ca2+ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca2+ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist‐evoked intracellular Ca2+.

Recruitment of Ca2+ channels by protein kinase C during rapid formation of putative neuropeptide release sites in isolated Aplysia neurons

Activation of protein kinase C (PKC) in Aplysia bag cell neurons causes the recruitment of voltage-dependent calcium channels. Using imaging techniques on isolated cells, we have now found that an activator of PKC, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), promotes the rapid appearance of new sites of calcium influx associated with a change in the morphology of neurite endings. In untreated cells, calcium influx triggered by action potentials occurs along neurites and in the central region of growth cones, but does not usually occur at the leading edge of lamellipodia. TPA produces extension of the lamellipodium, and action potentials now trigger calcium influx at the distal edge of the newly extended endings. Cotreatment with TPA and a cyclic AMP analog promotes movement of secretory organelles toward the new sites of calcium influx. Our results suggest that these second messenger systems promote the rapid formation of morphological structures that contribute to the potentiation of peptide release.

Giga-Ohm Seals on Intracellular Membranes: A Technique for Studying Intracellular Ion Channels in Intact Cells

A method is outlined for obtaining giga-ohm seals on intracellular membranes in intact cells. The technique employs a variant of the patch-clamp technique: a concentric electrode arrangement protects an inner patch pipette during penetration of the plasma membrane, after which a seal can be formed on an internal organelle membrane. Using this technique, successful recordings can be obtained with the same frequency as with conventional patch clamping. To localize the position of the pipette within cells, lipophilic fluorescent dyes are included in the pipette solution. These dyes stain the membrane of internal organelles during seal formation and can then be visualized by video-enhanced or confocal imaging. The method can detect channels activated by inositol trisphosphate, as well as other types of intracellular membrane ion channel activity, and should facilitate studies of internal membranes in intact neurons and other cell types.

Activation of a Ca2+-permeable cation channel produces a prolonged attenuation of intracellular Ca2+ release in Aplysia bag cell neurones

1 Brief synaptic stimulation, or exposure to Conus textile venom (CtVm), triggers an afterdischarge in the bag cell neurones of Aplysia. This is associated with an elevation of intracellular calcium ([Ca2+]i) through Ca2+ release from intracellular stores and Ca2+ entry through voltage‐gated Ca2+ channels and a non‐selective cation channel. The afterdischarge is followed by a prolonged (∼18 h) refractory period during which the ability of both electrical stimulation and CtVm to trigger afterdischarges or elevate [Ca2+]i is severely attenuated. By measuring the response of isolated cells to CtVm, we have now tested the contribution of different sources of Ca2+ elevation to the onset of the prolonged refractory period. 2 CtVm induced an increase in [Ca2+]i in both normal and Ca2+‐free saline, in part by liberating Ca2+ from a store sensitive to thapsigargin or cyclopiazonic acid, but not sensitive to heparin. 3 In the presence of extracellular Ca2+, the neurones became refractory to CtVm after a single application but recovered following ∼24 h, when CtVm could again elevate [Ca2+]i. However, this refractoriness did not develop if CtVm was applied in Ca2+‐free saline. Thus, elevation of [Ca2+]i alone does not induce refractoriness to CtVm‐induced [Ca2+]i elevation, but Ca2+ influx triggers this refractory‐like state. 4 CtVm produces a depolarization of isolated bag cell neurones. To determine if Ca2+ influx through voltage‐gated Ca2+ channels, activated during this depolarization, caused refractoriness to CtVm‐induced [Ca2+]i elevation, cells were depolarized with high external potassium (60 mm), which produced a large increase in [Ca2+]i. Nevertheless, subsequent exposure of the cells to CtVm produced a normal response, suggesting that Ca2+ influx through voltage‐gated Ca2+ channels does not induce refractoriness. 5 As a second test for the role of voltage‐gated Ca2+ channels, these channels were blocked with nifedipine. This drug failed to prevent the onset of refractoriness to CtVm‐induced [Ca2+]i elevation, providing further evidence that Ca2+ entry through voltage‐gated Ca2+ channels does not initiate refractoriness. 6 To examine if Ca2+ entry through the CtVm‐activated, non‐selective cation channel caused refractoriness, neurones were treated with a high concentration of TTX, which blocks the cation channel. TTX protected the neurones from the refractoriness to [Ca2+]i elevation produced by CtVm in Ca2+‐containing medium. 7 Using clusters of bag cell neurones in intact abdominal ganglia, we compared the ability of nifedipine and TTX to protect the cells from refractoriness to electrical stimulation. Normal, long‐lasting afterdischarges could be triggered by stimulation of an afferent input after a period of exposure to CtVm in the presence of TTX. In contrast, exposure to CtVm in the presence of nifedipine resulted in refractoriness. 8 Our data indicate that Ca2+ influx through the non‐selective cation channel renders cultured bag cell neurones refractory to repeated stimulation with CtVm. Moreover, the refractory period of the afterdischarge itself may also be initiated by Ca2+ entry through this cation channel.

Ca2+ influx and activation of a cation current are coupled to intracellular Ca2+ release in peptidergic neurons of Aplysia californica.

1. Stimulation of inputs to bag cell neurons in the abdominal ganglion of Aplysia californica causes an increase in their intracellular Ca2+ concentration ([Ca2+]i). We have used thapsigargin, a specific inhibitor of the endoplasmic reticulum Ca2+ pump, to analyse the effects of Ca2+ released from intracellular stores on the electrophysiological responses of bag cell neurons. 2. Using digital imaging of fura‐2‐loaded isolated bag cell neurons we found that thapsigargin rapidly evoked an increase in [Ca2+]i in somata, with smaller increases in neurites. Thapsigargin‐induced elevation of [Ca2+]i peaked at about 1 microM within 5‐10 min and then decayed to basal levels by 30 min. 3. Placement of an extracellular vibrating Ca(2+)‐selective microelectrode to within 1 micron of somata revealed a relatively large steady‐state Ca2+ efflux. Thapsigargin produced a rapid increase in Ca2+ influx. Changes in Ca2+ flux were not detected at neurites. 4. Thapsigargin produced a small depolarization in isolated bag cell neurons in artificial sea water (ASW). Sometimes enhanced depolarizations were observed when extracellular Na+ was replaced by TEA or Tris, but not N‐methyl‐D‐glucamine (NMDG). The depolarization was not blocked by 100 microM tetrodotoxin (TTX), removal of extracellular Ca2+ (0.5 mM EGTA) or addition of 10 mM Co2+ to the bath solution. 5. In voltage‐clamp experiments, thapsigargin induced an inward current (ITg) that was recorded in Ca(2+)‐free media containing TEA or Tris substituted for Na+. The apparent reversal potential of ITg was ‐16.8 +/‐ 1.2 mV in TEA‐ASW. Induction of ITg was inhibited in neurons that were microinjected with the Ca2+ chelator BAPTA‐Dextran70 or treated with the membrane‐permeant analogue BAPTA AM. Activation of ITg was not observed when Na+ was replaced with NMDG. Manipulation of [Na+]o and [K+]o produced shifts in the reversal potential of ITg consistent with the underlying channels being permeable to both Na+ and K+. 6. Thapsigargin did not alter the amplitude or kinetics of voltage‐activated Ba2+ currents, but in some experiments it did increase the amplitude of a component of outward K+ current. 7. Thapsigargin neither induced bag cell neurons within the intact ganglion to depolarize and fire spontaneously, nor did it alter the frequency or duration of firing of an electrically stimulated bag cell after‐discharge. 8. We conclude that thapsigargin‐sensitive Ca2+ pools are present predominantly in the somata of bag cell neurons. Ca2+ that is released from thapsigargin‐sensitive Ca2+ stores activates a non‐selective cation current that may help sustain depolarization of the somata, but does not by itself trigger an after‐discharge.

Dexamethasone-mediated inhibition of calcium transients and ACTH release in a pituitary cell line (AtT-20)

In the corticotroph-like murine pituitary tumor cell line, AtT-20, adrenocorticotropic hormone release is triggered by corticotropin-releasing hormone and is attenuated by the synthetic adrenal steroid dexamethasone. The precise mechanisms by which dexamethasone inhibits secretion are under investigation. We examined whether dexamethasone can modulate release via regulation of calcium homeostasis. More specifically, we have evaluated the effects of dexamethasone on calcium current, intracellular calcium concentration, and adrenocorticotropic hormone release. Using perforated patch-clamp and calcium imaging with fura PE3/AM, we found that dexamethasone decreases calcium current and intracellular calcium levels. The inhibition of current by dexamethasone is not, however, altered by the calcium channel antagonists nifedipine (L-type) or omega-agatoxin IVA (P/Q-type), despite the presence of these calcium channel subtypes in AtT-20 cells and the exclusive coupling of adrenocorticotropic hormone release to the L-type channel in these cells. We also evaluated the temporal relationship between dexamethasone-mediated inhibition of secretion and calcium influx. Whereas a prolonged (2 h) incubation with dexamethasone inhibits corticotropin-induced release by approximately 40%, a rapid (10 min) incubation (a time interval sufficient for dexamethasone-mediated inhibition of calcium transients) does not inhibit release. These data suggest, therefore, that dexamethasone does, indeed, modulate calcium homeostasis in AtT-20 cells, but that this effect is not responsible for its inhibition of secretion.

Hyperosmotic media inhibit voltage-dependent calcium influx and peptide release in Aplysia neurons

SummaryThe bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20–30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.