Disrupted [Ca2+]i homeostasis contributes to the toxicity of nitric oxide in cultured hippocampal neurons

Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+]i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca2+]i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S-nitrosocysteine (SNOC; 300 microM) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+]i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20-min SNOC treatment failed to prevent the late [Ca2+]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca2+]i homeostatic mechanisms, and that the resulting persistent [Ca2+]i elevations contribute to the delayed neurotoxicity of NO.     

Expression of mRNAs encoding dopamine receptors in striatal regions is differentially regulated by midbrain and hippocampal neurons

The glutamate analogue kainic acid was injected into the hippocampus of intact or 6-hydroxydopamine deafferented rats to investigate the influence of hippocampal neurons on the expression of dopamine D1 and D2 receptor mRNAs in subregions of the striatal complex and possible modulation by dopaminergic neurons. Quantitative in situ hybridization using 35S-labeled oligonucleotide probes specific for dopamine D1 and D2 receptor mRNAs, respectively, were used. It was found that an injection of kainic acid into the hippocampal formation had alone no significant effect on dopamine D1 or D2 receptor mRNA levels in any of the analyzed striatal subregions in animals analyzed 4 h after the injections. Kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion produced an increase in D1 receptor mRNA levels in the ipsilateral medial caudate-putamen, and a bilateral increase in core and shell of nucleus accumbens (ventral striatal limbic regions). A unilateral 6-hydroxydopamine lesion alone caused an increase in D2 receptor mRNA in the lateral caudate-putamen (dorsal striatal motor region) ipsilateral to the lesion and an increase in D1 receptor mRNA in the accumbens core ipsilateral to the lesion. However, in dopamine-lesioned animals, dopamine D1 receptor mRNA levels were increased bilaterally in nucleus accumbens core and shell and in the ipsilateral medial caudate-putamen following kainic acid stimulation in the hippocampus ipsilateral to the dopamine lesion. These results indicate a differential regulation of the expression of dopamine D1 and D2 receptor mRNAs by midbrain and hippocampal neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dendritic arbor of locus coeruleus neurons contributes to opioid inhibition

1. The nucleus locus coeruleus (LC) is made up of noradrenergic cells all of which are hyperpolarized by opioids. Recent work has shown that the reversal potential of the opioid-induced current is more negative than the potassium equilibrium potential. The aim of the present study was to determine whether the extent of the dendritic field could contribute to the very negative opioid reversal potential. 2. Individual LC cells were labeled in the brain slice preparation. The number of dendrites found on cells in slices sectioned in the horizontal plane was greater than cells in coronal slices. However, the dimensions of the cell body slices from each plane were not significantly different. 3. The resting conductance of neurons from slices cut in the horizontal plane was significantly larger than in cells from coronal plane. 4. The amplitude of the outward current induced by [Met5]-enkephalin (ME) was larger in cells from horizontal slices and the reversal potential was more negative than that of cells in coronal slices. 5. The results show that the plane of section influences the membrane properties and opioid actions of LC neurons in vitro and suggest that these differences correlate with the numbers of dendrites. The results suggest that in vivo, in addition to intrinsic membrane properties and synaptic inputs, the structural makeup of the nucleus is an important factor in determining the activity.     

Microtubule transport from the cell body into the axons of growing neurons

The present studies test the hypothesis that microtubules (MTs) are transported from the cell body into the axons of growing neurons. Dissociated sympathetic neurons were cultured using conditions that allow us to control the initiation of axon outgrowth. Neurons were injected with biotin-labeled tubulin (Bt-tub) and then stimulated to extend axons. The newly formed axons were then examined using immunofluorescence procedures for MTs with or without Bt-tub. Because the Bt-tub is fully assembly competent, all MTs that assemble after injection will contain Bt-tub. However, MTs that exist in the neuron at the time of injection and persist during the subsequent incubation will not contain Bt-tub. Because the neurons were injected before extending axons, MTs without Bt-tub are initially localized to the cell body. We specifically determined whether these MTs appeared in the newly formed axon. Such a result can only be explained by the transport of these MTs from their initial location in the cell body into the axon. The newly formed axons of many neurons contained MTs both with and without Bt-tub. MTs without Bt-tub were detected all along the axon and in some neurons represented a substantial portion of the total polymer in the proximal and middle regions of the axon. These results show that MTs are transported from the cell body into growing axons and that this transport is robust, delivering MTs to all regions of the newly formed axon.     

Ultrastructural localization of actin in the cell body of rat spinal ganglion neurons

We used phalloidin staining and immunocytochemistry at the light and electron microscope level to determine the localization of actin in the cell bodies of rat spinal ganglion neurons. The results show that actin is mostly concentrated along the periphery of the neuronal perikaryon, including the perikaryal projections. This localization places actin in a strategic position to be influenced by incoming signals and to produce mechanical tensions able to shape the perikaryal surface.     

Programmed cell death contributes to postnatal lung development

The rat lung undergoes the phase of maturation of the alveolar septa and of the parenchymal microvascular network mainly during the third postnatal week. Speculating that programmed cell death may contribute to the thinning of the alveolar septa, we searched for the presence of DNA fragmentation in rat lungs between postnatal days 6 and 36 using the TUNEL procedure. The number of positive nuclei was compared at different days. We observed an 8-fold increase of programmed cell death toward the end of the third week as compared to the days before and after this time point. The precise timing of the appearance of the peak depended on the size of the litter. Double-labeling for DNA fragmentation (TUNEL) and for type I and type II epithelial cells (antibodies E11 and MNF-116), as well as morphologic studies at electron microscopic level, revealed that during the peak of programmed cell death mainly fibroblasts and type II epithelial cells were dying. While both dying cell types were TUNEL-positive, nuclear fragments and apoptotic bodies were exclusively observed in the dying fibroblasts. We conclude that programmed cell death is involved in the structural maturation of the lung by reducing the number of fibroblasts and type II epithelial cells in the third postnatal week. We observed that the dying fibroblasts are cleared by neighboring fibroblasts in a later stage of apoptosis, and we hypothesize that type II epithelial cells are cleared by alveolar macrophages in early stages of the programmed cell death process.     

An ultrastructural study of the hippocampal neurons in mice subjected to heat exposure

The ultrastructure of hippocampal neurons in mice subjected to heat treatment for 40 min at 42 degrees C was studied. Such a treatment has been shown to result in significant changes in the structure of pyramidal neurons. An increased chromatin condensation in the nuclei was found in addition to deep invagination of their envelopes and reduction or fragmentation of the reticulum cisterns. These changes were accompanied by mitochondrial swelling and increased number of clathrine vesicles around the Golgi complex. The heat exposure followed by feeding on vitamins and beta-carotin appeared to diminish chromatin condensation and kept the cisterns invariable.     

Duration of neurofibrillary changes in the hippocampal pyramidal neurons

The clinical course of grave forms of leptospirosis presents with disorders in the fluid and electrolyte balance and acid-base condition (ABC) which fact necessitates taking prompt action for the condition to be corrected. Correction of disorders in the fluid and electrolyte balance involves employment of glucose and salt solutions, dextrans, and in most severe cases albumin drugs under control of hematocrit values, plasma osmolarity, and 24-h diuresis monitoring. Correction of disorders in the ABC is primarily aimed at alleviating the metabolic acidosis through detoxication by applying specific therapy together with oral and parenteral administration of sodium hydrogen carbonate.     

Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway

The expression of tissue plasminogen activator (tPA) is increased during activity-dependent forms of synaptic plasticity. We have found that inhibitors of tPA inhibit the late phase of long-term potentiation (L-LTP) induced by either forskolin or tetanic stimulation in the hippocampal mossy fiber and Schaffer collateral pathways. Moreover, application of tPA enhances L-LTP induced by a single tetanus. Exposure of granule cells in culture to forskolin results in secretion of tPA, elongation of mossy fiber axons, and formation of new, active presynaptic varicosities contiguous to dendritic clusters of the glutamate receptor R1. These structural changes are blocked by tPA inhibitors and induced by application of tPA. Thus, tPA may be critically involved in the production of L-LTP and specifically in synaptic growth.     

Nicotine modifies the activity of ventral tegmental area dopaminergic neurons and hippocampal GABAergic neurons

While trying to mimic the dose and time course of nicotine as it is obtained by a smoker, we found the following results. The initial arrival of even a low concentration of nicotine increased the firing rate of dopaminergic neurons from the ventral tegmental area (VTA) and increased the spontaneous vesicular release of GABA from hippocampal neurons. Longer exposure to nicotine caused variable, but dramatic, desensitization of nicotinic receptors and diminished the effects of nicotine. The addictive properties of nicotine as well as its diverse effects on cognitive function could be mediated through differences in activation and desensitization of nicotinic receptors in various areas of the brain.     

Slow binding of phenytoin to inactivated sodium channels in rat hippocampal neurons

The anticonvulsant phenytoin inhibited Na+ currents in rat hippocampal neurons with a potency that increased dramatically at depolarized holding potentials, suggesting weak binding to resting Na+ channels but tight binding to open or inactivated channels. Four different experimental measurements, i.e., steady block at different holding potentials, on and off kinetics at depolarized holding potentials, shifts in the inactivation curve, and dose-dependent slowing of recovery from inactivation, yielded an estimated Kd of approximately 7 microM for phenytoin binding to inactivated channels. Prolonged depolarizations of at least several seconds were necessary for significant block by therapeutic concentrations of phenytoin. The slow development of block does not reflect selective binding of phenytoin to slow inactivated states of the channel, because block developed faster and required less depolarized voltages than did slow inactivation. Instead, it appears that phenytoin binds tightly but slowly (approximately 10(4) M-1 sec-1) to fast inactivated states of the Na+ channels. This tight but slow binding may underlie the ability of phenytoin to disrupt epileptic discharges with minimal effects on normal firing patterns.     

Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called beta-amyloid (Abeta) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Abeta that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-beta-cyclodextrin, we show that the formation of Abeta is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Abeta formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Abeta formation to occur and imply a link between cholesterol, Abeta, and Alzheimer's disease.     

Calcium-induced activation of the mitochondrial permeability transition in hippocampal neurons

The mitochondrial permeability transition (mPT) has been implicated in both excitotoxic and apoptotic neuronal cell death, despite the fact that it has not been previously identified in neurons. To study the mPT in hippocampal neurons, cultures were loaded with the mitochondrial dye JC-1 and observed with confocal and conventional microscopy. After pretreatment with 4Br-A23187 and subsequent calcium addition, the initially rodlike mitochondria increased in diameter until mitochondria became rounded in appearance. Morphological changes reversed when calcium was removed by EGTA. When neurons were loaded with both fura-2-AM and rhodamine 123, calcium loading produced an increase in cytosolic calcium, mitochondrial depolarization, and similar alterations in mitochondrial morphology. Smaller calcium challenges produced calcium cycling, delaying morphological changes until after secondary depolarization and calcium release to the cytosol. In neurons exposed to glutamate, confocal observation of JC-1 fluorescence revealed comparable changes in mitochondrial morphology that were prevented when barium was substituted for calcium, or following pretreatment with the mPT inhibitor, cyclosporin A. These experiments establish conditions in which the mPT could be observed in situ in neurons in response to calcium loading. In addition, the timing of changes suggested that induction of the permeability transition in situ represents a sequence of multiple events that may reflect the multiple open conformations of the mPT pore.     

Selective reduction of GluR2 protein in adult hippocampal CA3 neurons following status epilepticus but prior to cell loss

Kainic acid (KA) induces status epilepticus and delayed neurodegeneration of CA3 hippocampal neurons. Downregulation of glutamate receptor 2 (GluR2) subunit mRNA [the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) subunit that limits Ca2+ permeability] is thought to a play role in this neurodegeneration, possibly by increased formation of Ca2+ permeable AMPA receptors. The present study examined early hippocampal decreases in GluR2 mRNA and protein following kainate-induced status epilepticus and correlated expression changes with the appearance of dead or dying cells by several histological procedures. At 12 h, in situ hybridization followed by emulsion dipping showed nonuniform decreases in GluR2 mRNA hybridization grains overlying morphologically healthy-appearing CA3 neurons. GluR1 and N-methyl-D-aspartate receptor mRNAs were unchanged. At 12-16 h, when little argyrophilia or cells with some features of apoptosis were detected by silver impregnation or electron microscopy, single immunohistochemistry with GluR2 and GluR2/3 subunit-specific antibodies demonstrated a pattern of decreased GluR2 receptor protein within CA3 neurons that appeared to predict a pattern of damage, similar to the mRNA observations. Double immunolabeling showed that GluR2 immunofluorescence was depleted and that GluR1 immunofluorescence was sustained in clusters of the same CA3 neurons. Quantitation of Western blots showed increased GluR1:GluR2 ratios in CA3 but not in CA1 or dentate gyrus subfields. Findings indicate that the GluR1:GluR2 protein ratio is increased in a population of CA3 neurons prior to significant cell loss. Data are consistent with the "GluR2 hypothesis" that reduced expression of GluR2 subunits will increase formation of AMPA receptors permeable to Ca2+ and predict vulnerability to a particular subset of pyramidal neurons following status epilepticus.     

Functional expression of TrkA receptors in hippocampal neurons

Nerve growth factor (NGF) initiates its biological effects by promoting the dimerization and activation of the tyrosine kinase receptor TrkA. The requirements for NGF signaling through the TrkA receptor have been defined extensively from studies in immortalized cells, involving transfection of NIH 3T3, COS, and PC12 cells. In the present study, we tested the effects of extracellular and intracellular mutations of TrkA after DNA-mediated transfection in primary cultures of embryonic day 17 hippocampal neurons. We found that the action of the TrkA receptor on neuronal differentiation depends on specific motifs in the extracellular domain and on tyrosine 490 (Y490), the site for SHC protein binding. In contrast with previous observations in a PC12 background, a mutation in the SHC Y490 binding site in TrkA resulted in a loss of NGF-dependent process formation. These results indicate that tyrosine 490 is necessary for neurite outgrowth in hippocampal neurons. Moreover, a constitutively active form of TrkA did not give enhanced responsiveness in hippocampal neurons, indicating that the behavior of TrkA receptors in primary neuronal cells is distinct from that of other cell types.     

Amyloid beta-protein impairs astrocyte-mediated differentiation of hippocampal neurons

Embryonic rat hippocampal neurons were cultured on poly-D-lysine (PDL) or on cortical astrocytes, some of which had been pretreated for 24 h with amyloid beta-protein (beta-AP). Amino acid-induced currents were quantified. Membrane capacitance (Cm), as well as the amplitude and density of amino acid-evoked currents recorded in neurons cultured on untreated astrocytes were all statistically greater than those recorded in neurons grown on PDL. However, compared to untreated astrocytes, those treated with beta-AP led to significantly lower values in neurons for Cm and GABA, kainate- and NMDA-induced currents, while glycine-activated current values were not significantly different. Furthermore, beta-AP treatment abolished spontaneous Cac2+ fluctuations in astrocytes, which may account for their impaired ability to promote the expression of functional transmitter receptors in neurons.     

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons

The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.     

The alpha subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons

Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.