Library

Notes: Abstract: In the present study we describe an ELISA to quantify the light subunit of the neurofilament triplet protein (NFL) in CSF. The method was validated by measuring CSF NFL concentrations in healthy individuals and in two well-characterized groups of patients with amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). The levels were increased in ALS (1,743 ± 1,661 ng/L; mean ± SD) and AD (346 ± 176 ng/L) compared with controls (138 ± 31 ng/L; p 〈 0.0001 for both). Within the ALS group, patients with lower motor neuron signs only had lower NFL levels (360 ± 237 ng/L) than those with signs of upper motor neuron disease (2,435 ± 1,633 ng/L) (p 〈 0.05). In a second study patients with miscellaneous neurodegenerative diseases were investigated (vascular dementia, olivopontocerebellar atrophy, normal pressure hydrocephalus, cerebral infarctions, and multiple sclerosis), and the CSF NFL level was found to be increased (665 ± 385 ng/L; p 〈 0.0001). NFL is a main structural protein of axons, and we suggest that CSF NFL can be used to monitor neurodegeneration in general, but particularly in ALS with involvement of the pyramidal tract.

Notes: Abstract: We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age-related changes in calpain specificity and substrate susceptibility were measured. Several age-related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).

Notes: Abstract: The effect of hydrocephalus on cerebral energy metabolites and on intermediates of membrane phospholipid metabolism has been studied in H-Tx rats with inherited infantile hydrocephalus. Hydrocephalic rats and rats with shunts placed at 4–5 days or at 10 days after birth were subjected to magnetic resonance imaging in vivo before 21 days of age to determine the dimensions of the ventricles and cortex. At 21 days, the brains from the three groups of rats, together with age-matched control littermates, were frozen in situ, and chloroform/methanol extracts of cerebral cortex were prepared for high-resolution 31P-NMR spectroscopy. Hydrocephalus resulted in modest decreases in most metabolites quantified. Levels of phosphocreatine, ATP, and diphosphodiesters plus NAD were significantly reduced by 23–32%, and inorganic phosphate content was reduced but not significantly. Levels of the membrane phospholipid intermediates phosphorylethanolamine, glycerophosphorylethanolamine, and glycerophosphorylcholine were also significantly reduced by 30–33%, indicating changes in membrane metabolism. These general decreases are consistent with a loss of cell contents, possibly due to changes in dendrite structure in hydrocephalus. Rats shunt-treated at 4–5 days were similar to control rats for all energy metabolites, but those treated later at 10 days had reduced phosphocreatine and ATP levels. Shunt-treated rats also had reductions in levels of membrane phospholipids, some of which occurred in sham-operated rats. It is concluded that hydrocephalus leads to reductions in levels of energy metabolites and in levels of membrane phospholipids and that the changes in energy metabolites can be reversed by early, but not by later, shunt treatment.

Notes: Abstract: We used in vitro translation and antibodies against phosphoserine and the eukaryotic initiation factors eIF-4E, eIF-4G, and eIF-2α to examine the effects of global brain ischemia and reperfusion on translation initiation and its regulation in a rat model of 10 min of cardiac arrest followed by resuscitation and 90 min of reperfusion. Translation reactions were performed on postmitochondrial supernatants from brain homogenates with and without aurintricarboxylic acid to separate incorporation due to run-off from incorporation due to peptide synthesis initiated in vitro. The rate of leucine incorporation due to in vitro-initiated protein synthesis in normal forebrain homogenates was ∼0.4 fmol of leucine/min/µg of protein and was unaffected by 10 min of cardiac arrest, but 90 min of reperfusion reduced this rate 83%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blots of these homogenates showed that neither 10 min of global brain ischemia nor 90 min of reperfusion induced significant alterations in the quantity or serine phosphorylation of eIF-4E. However, we observed in all 90-min-reperfused samples eIF-4G fragments that also bound eIF-4E. The amount of eIF-2α was not altered by ischemia or reperfusion, and immunoblotting after isoelectric focusing did not detect serine-phosphorylated eIF-2α in normal samples or in those obtained after ischemia without reperfusion. However, serine-phosphorylated eIF-2α was uniformly present after 90 min of reperfusion and represented 24 ± 3% of the eIF-2α in these samples. The serine phosphorylation of eIF-2α and partial fragmentation of eIF-4G observed after 90 min of reperfusion offer an explanation for the inhibition of protein synthesis.

Notes: Abstract: The protease inhibitor α1-antichymotrypsin (ACT) has been suggested to be involved in the etiology of Alzheimer's disease (AD). Increased levels of ACT have been found in serum and brains of AD patients, and ACT has been proposed to regulate β-amyloid fibril formation in vitro. To gain insight into the regulation of ACT in the brain, we investigated the signal transduction pathways involved in ACT gene expression and protein synthesis in the human astrocytoma cell line U373. This cell line has previously been shown to respond with strong ACT synthesis on stimulation with interleukin-1β (IL-1β) or tumor necrosis factor-α (TNFα). Here, we describe that both IL-1β and TNFα activate the transcription factor nuclear factor-κB (NF-κB) via production of reactive oxygen intermediates resulting in ACT expression. In addition, we show that neither protein kinase C nor protein kinase A is involved in IL-1β- or TNFα-induced ACT expression. These results suggest that activation of NF-κB may be one possible cause of increased ACT levels in AD and provide a basis for the development of drugs used for the modulation of inflammatory processes occurring in AD.

Notes: Abstract: Hyperoxia has been considered a model of free radical reactive oxygen species production in aging and age-related disorders. Previously, we studied the membrane protein alterations that occur during hyperoxia; we found that exposure of young animals to 24 h of hyperoxia provided the greatest degree of oxidation of cortical synaptosomal membrane proteins. We reasoned that free radical oxidation was involved in this protein oxidation. In accordance, in the current study we investigated the protective nature of two known free radical scavengers, N-tert-butyl-α-phenylnitrone (PBN) and 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Tempol), against 24-h hyperoxia damage. The three techniques used in this study were electron paramagnetic resonance (EPR) protein-specific spin labeling, assay of the activity of the oxidatively sensitive enzyme glutamine synthetase (GS), and measurement of protein carbonyl content. Before hyperoxia, gerbils received intraperitoneal injections of varying concentrations of either of the two free radical scavengers. After 30 min, the gerbils were exposed to 90–100% O2 for 24 h. For the spin labeling experiments, cortical synaptosomes were isolated from gerbils. The membrane proteins were spin labeled with the thiol-specific label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidin-1-oxyl). As in our earlier study, the EPR spectral parameter of MAL-6-labeled membranes, the W/S ratio, decreased with hyperoxia (p 〈 0.00001). This effect was lessened significantly with administration of PBN (p 〈 0.0003) or Tempol (p 〈 0.00003). For the GS and protein carbonyl assays, cortical proteins were used. The activity of the GS decreased with hyperoxia (p 〈 0.000005), and this effect likewise was lessened with administration of PBN (p 〈 0.004) or Tempol (p 〈 0.002). The protein carbonyl content increased with hyperoxia (p 〈 0.0002), and there was a protective effect found with Tempol (p 〈 0.000001). The optimum doses for PBN and Tempol were 20 and 5 mg/kg, respectively. The results are discussed with reference to the use of free radical scavengers as potential antiaging agents.

Notes: Abstract: Preliminary evidence suggests adenosine, a neuromodulator, has neuroprotective properties during cerebral ischemia. It is unclear, however, if adenosine has glioprotective effects. We studied the effect of adenosine on cellular injury in astroglial cultures subjected to combined glucose-oxygen deprivation. Adenosine (100–1,000 µM) dramatically reduced astroglial injury, whereas the adenosine agonists 2-chloroadenosine (10 nM–100 µM), N6-cyclopentyladenosine (1 nM–10 µM), 5′-N-ethylcarboxamidoadenosine (10 nM–100 µM), and N6-2-(4-aminophenyl)ethyladenosine (10 nM–100 µM) had no effect. Furthermore, the adenosine antagonists 8-cyclopentyl-1,3-dipropylxanthine (1 nM–1 µM), xanthine amine congener (10 nM–10 µM), and 8-(p-sulfophenyl)-theophylline (10–300 µM) failed to reverse the protective effect of 200 µM adenosine. Next, adenosine degradation products were studied. Inosine proved to be glioprotective at concentrations nearly identical to those of adenosine, but hypoxanthine and ribose had no effect. The protective effect of 200 µM inosine was not reversed by 8-(p-sulfophenyl)theophylline (10–300 µM). Adenosine deaminase (1 unit/ml) had no effect on protection produced by adenosine, whereas erythro-9-(2-hydroxy-3-nonyl)adenine hydrochloride (10 µM) reversed the protective effect of adenosine. Dipyridamole (4 µM) inhibited the protective effect of both adenosine and inosine. We conclude that adenosine dramatically decreases astroglial injury during combined glucose-oxygen deprivation and that this protective effect appears to be mediated by inosine.

Notes: Abstract: Cytochrome P450 2E1 (CYP2E1) metabolizes several neuroactive substrates, including exogenous compounds such as anesthetics, organic solvents, and muscle relaxants as well as endogenous substrates such as arachidonic acid. CYP2E1 and its mRNA were found to be expressed in the rat hippocampus, where the enzyme was localized mainly to the microsomal fraction. Chlorzoxazone (CZN), a CYP2E1 substrate, was 6-hydroxylated in hippocampal homogenates with a Km of 25.5 µM and a Vmax of 0.22 pmol/mg/min. CYP2E1 was also expressed in vitro in cortical glial cultures, where CYP2E1 mRNA levels were found to be 1,000-fold lower than in rat liver. Exposure of cortical glial cultures to 25 or 100 mM ethanol for 24 h caused a fourfold and sixfold increase, respectively, in the rate of CYP2E1-dependent 6-hydroxylation of CZN. After a continuous exposure to 100 mM ethanol for 48 or 72 h, however, the hydroxylation rate was down-regulated. Chlormethiazole, a potent inhibitor of hepatic CYP2E1 transcription, inhibited the ethanol-dependent induction of CYP2E1 by 50%. In vivo, acute ethanol treatment of rats (24 h, 3 g/kg) resulted in a 1.8-fold increase in the rate of CZN 6-hydroxylation in hippocampal homogenates. It is concluded that CYP2E1 is expressed and catalytically active in the rat CNS, and that CYP2E1 can be induced by a relatively low concentration of ethanol in cortical glial cultures. It is suggested that CYP2E1 substrates may be metabolically activated in situ in the CNS.

Notes: Abstract: Amyloid β protein (Aβ), which accumulates in the senile plaques in the brain of Alzheimer's patients, is cytotoxic to neurons. A modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, in which a yellow redox dye, MTT, is reduced to purple formazan, is very sensitive to the effect of Aβ. In primary hippocampal cultures, inhibition of MTT reduction starts within 2 h after the addition of low concentrations of Aβ and reaches a plateau in 12 h. This effect of Aβ is not blocked by Ca2+ channel blockers or in Ca2+-free medium. In contrast, lactate dehydrogenase (LDH) release and trypan blue exclusion, which are indices of cell death, start 3 days after exposure to high concentrations of Aβ and are blocked by Ca2+ channel blockers such as Co2+, nicardipine, and diltiazem. When Aβ was washed out from the medium after 12 h, MTT reduction recovers and LDH release does not occur, suggesting that a long-lasting inhibition of the cellular redox system may be required to induce cell death. These observations demonstrate that Aβ toxicity consists of two phases—a Ca2+-independent early phase and a Ca2+-dependent late phase—and that the early phase may be required to induce the late phase.

Notes: Abstract: The in vivo presence of tryptophan hydroxylase activity in rat major cerebral arteries as well as the possible origin of the structure containing it were explored. Enzyme activity was appraised by accumulation of 5-hydroxytryptophan after inhibition of aromatic l-amino acid decarboxylase. Decarboxylase inhibition evoked a significant increase in 5-hydroxytryptophan levels in rat cerebral arteries, striatum, hippocampus, hypothalamus, and plasma but had no effect on aorta. p-Chlorophenylalanine reduced 5-hydroxytryptophan accumulation in the cerebral vessels and brain nuclei, whereas α-methyltyrosine did not modify it except in hypothalamus, where it was enhanced. α-Methyltyrosine significantly reduced noradrenaline levels in cerebral arteries and l-dopa accumulation after inhibition of the decarboxylase in striatum. Dorsal raphe nucleus lesioning significantly diminished 5-hydroxytryptophan formation in cerebral arteries, striatum, and hypothalamus, without affecting it in hippocampus. Lesion of median raphe nucleus reduced 5-hydroxytryptophan accumulation in hippocampus and in hypothalamus but not in cerebral blood vessels or striatum. Superior cervical ganglia removal decreased noradrenaline levels in cerebral blood vessels without affecting 5-hydroxytryptophan accumulation. These results indicate the presence of a functionally active tryptophan hydroxylase in rat cerebral arteries associated with fibers originating from dorsal raphe nucleus. This supports that rat major cerebral arteries receive serotonergic innervation from central origin.

Notes: Abstract: The anticonvulsant carbamazepine is an effective treatment both for epilepsy and for bipolar affective disorder, but the molecular mechanism(s) underlying its therapeutic effects have not been identified. We have found that carbamazepine exerts significant inhibitory effects on the cyclic AMP (cAMP) generating system. Within the clinical therapeutic range (∼50 µM), carbamazepine inhibited both basal and forskolin-stimulated cAMP production, without having any significant effects on phosphodiesterase activity. Carbamazepine also exerted its inhibitory effects on the cAMP generating system in pertussis toxin-treated cells, suggesting that the action of carbamazepine was likely mediated through an inhibitory guanine nucleotide binding protein-independent mechanism. A forskolin affinity purification column was used to purify adenylyl cyclases from rat cerebral cortex, and we found that carbamazepine inhibited both basal and forskolin-stimulated activity of purified adenylyl cyclase. We also investigated the effects of carbamazepine on the levels of the transcription factor, cAMP response element binding protein in the phosphorylated (active) state, and found that carbamazepine significantly inhibited forskolin-induced phosphorylation of the cAMP response element binding protein. The data indicate that carbamazepine inhibits adenylyl cyclase activity as well as the downstream effects of activation of adenylyl cyclase.

Notes: Abstract: Aromatase in the diencephalic neurons, the level of which increases transiently during the prenatal to neonatal period, has been suggested to be involved in control of sexual behavior and differentiation of the CNS. Effects of neurotransmitters on levels of aromatase mRNA in cultured neurons were investigated to determine factors regulating the developmental increase that occurs in level of fetal brain aromatase. The expression of aromatase in diencephalic neurons of fetal mice at embryonic day 13, cultured in vitro, was significantly affected by α1-adrenergic receptor ligands. Aromatase mRNA levels were higher in neurons treated with the α1-agonist phenylephrine than in control neurons, whereas prazosin, an α1-antagonist, suppressed this increase, and ligands for α2- or β-adrenergic receptors did not exert any influence. The profile of α1-adrenergic receptor subtypes during actual development in vivo suggested that the α1B subtype is in fact responsible for the signal transduction. Substance P, cholecystokinin, neurotensin, and brain natriuretic peptide also increased the level of expression along with phorbol 12-myristate 13-acetate and dibutyryl-cyclic GMP, whereas forskolin and dibutyryl-cyclic AMP caused a decrease. These data indicate that stimulation via α1 (possibly α1B)-adrenergic receptors, as well as receptors of specific neuropeptides, controls the expression of aromatase in embryonic day 13 diencephalic neurons through activation of protein kinase C or G. β-Adrenergic receptors would not appear to participate in the regulation, judging from their developmental profile, although cyclic AMP might be a suppressive second messenger.

Notes: Abstract: β-Amyloid protein has been implicated as a potential causative agent in the neuropathology associated with Alzheimer's disease. This possibility is supported by observations that β-amyloid induces neuronal degeneration and astrocyte reactivity in vitro by as yet undefined mechanism(s). In this report, we present data demonstrating that the pathological effects of β-amyloid on cultured cells are modulated by activation of the thrombin receptor. At concentrations between 50 and 500 nM, thrombin pretreatment significantly attenuates neurotoxicity mediated by fibrillar aggregates of β1–42 and β25–35 peptides. In cultured astrocytes, the stellate morphology induced by β1–42 and β25–35 aggregates can be prevented and reversed by thrombin exposures between 10 pM and 1 µM. In contrast, thrombin potentiates rather than attenuates the β-amyloid-induced increased expression of basic fibroblast growth factor, suggesting that thrombin differentially modulates the effects of β-amyloid on astrocytes. Thrombin's effects on both neurons and astrocytes are mimicked by thrombin receptor-activating peptide and inhibited by two potent thrombin inhibitors, hirudin and protease nexin-1. These data provide both new insight into the signaling pathways underlying the cellular effects of β-amyloid and additional support for the role of thrombin as an important mediator of neuropathological events.

Notes: Abstract: To elucidate mechanisms regulating the production of platelet-derived growth factor (PDGF) in the CNS, we analyzed the influence of a panel of cytokines on PDGF mRNA and protein levels in astrocyte-enriched cultures from the human embryonic brain and spinal cord. Using a specific ELISA, PDGF AB protein was detected in serum-free astrocyte supernatants and its levels were significantly increased after treatment of the cultures with transforming growth factor-β1 (TGF-β1) or tumor necrosis factor-α (TNF-α); the largest increase was detected after combined treatment with the two cytokines. Interleukin-1β (IL-1β) by itself had little or no effect but synergized with TGF-β1 in enhancing PDGF AB production. Supernatants from human astrocyte cultures stimulated the proliferation of rat oligodendrocyte progenitors, and most of the mitogenic activity could be accounted for by PDGF. By northern blot analysis, both PDGF A- and PDGF B-chain mRNAs were detected in untreated astrocytes. PDGF B-chain mRNA levels were increased by TGF-β1, TNF-α, TNF-α/TGF-β1, or IL-1β/TGF-β1, whereas PDGF A-chain mRNA levels were not consistently affected by cytokine treatments. These in vitro data indicate that TGF-β1, TNF-α, and IL-1β are able to stimulate astrocyte PDGF production. This cytokine network could play a role in CNS development and repair after injury or inflammation.

Notes: Abstract: δ-Opioids mobilize Ca2+ from intracellular stores in undifferentiated NG108-15 cells, but the mechanism involved remains unclear. Therefore, we examined the effect of [d-Pen2,5]enkephalin on inositol 1,4,5-trisphosphate formation in these cells. [d-Pen2,5]enkephalin caused a dose-dependent (EC50 = 3.1 nM) increase in inositol 1,4,5-trisphosphate formation (measured using a specific radioreceptor mass assay), which peaked (25.7 ± 1.2 pmol/mg of protein with 1 µM, n = 9) at 30 s and returned to basal levels (10.6 ± 0.9 pmol/mg of protein, n = 9) within 4–5 min. This response was fully naloxone (1 µM) reversible and pertussis toxin (100 ng/ml for 24 h) sensitive. Preincubation with Ni2+ (2.5 mM) or nifedipine (1 µM) had no effect on the [d-Pen2,5]enkephalin (1 µM)-induced inositol 1,4,5-trisphosphate response, and K+ (80 mM) was unable to stimulate inositol 1,4,5-trisphosphate formation, indicating Ca2+ influx-induced activation of phospholipase C is not involved. Preincubation with the protein kinase C inhibitor Ro 31-8220 (1 µM) enhanced, whereas acute exposure to phorbol 12,13-dibutyrate (1 µM) abolished, the [d-Pen2,5]enkephalin (0.1 µM)-induced inositol 1,4,5-trisphosphate response, suggesting protein kinase C exerts an autoinhibitory feedback action. [d-Pen2,5]Enkephalin also dose-dependently (EC50 = 2.8 nM) increased the intracellular [Ca2+], which was maximal (24 nM increase with 1 µM, n = 5) at 30 s. This close temporal and dose-response relationship strongly suggests that δ-opioid receptor-mediated increases in intracellular [Ca2+] results from inositol 1,4,5-trisphosphate-induced Ca2+ release from intracellular stores, in undifferentiated NG108-15 cells.

Notes: Abstract: The G proteins Gs and Gi1 appear to be capable of binding to tubulin specifically, and it has been suggested that such binding results in G protein activation via direct transfer of GTP. This study was undertaken to demonstrate that consequences of G protein activation by tubulin, i.e., stimulation or inhibition of adenylyl cyclase, were dependent on the G proteins expressed as well as unique aspects of the membrane or cytoskeleton in a given cell type. Membranes from rat C6 glioma cells, which express Gsα but not Giα1, responded to the addition of tubulin with a stable activation of adenylyl cyclase. Conversely, membranes from rat cerebral cortex, which contain both Gs and Gi1, responded to exogenous tubulin with a stable inhibition of adenylyl cyclase. Unlike C6 membranes, cerebral cortex membranes are richly endowed with tubulin, and antitubulin antibodies immunoprecipitated complexes of tubulin and Gi1 or Gs from detergent extracts of these membranes. Nearly 90% of the Gsα from Triton X-114 extracts coimmunoprecipitated with tubulin, suggesting that these proteins exist as a complex in the synaptic membrane. Such complexes may provide the framework for a G protein-cytoskeleton link that participates in the modulation of cellular signal transduction.

Notes: Abstract: Dystroglycan is encoded by a single gene and cleaved into two proteins, α- and β-dystroglycan, by posttranslational processing. The 120-kDa peripheral nerve isoform of α-dystroglycan binds laminin-2 comprised of the α2, β1, and γ1 chains. In congenital muscular dystrophy and dy mice deficient in laminin α2 chain, peripheral myelination is disturbed, suggesting a role for the dystroglycan-laminin interaction in peripheral myelinogenesis. To begin to test this hypothesis, we have characterized the dystroglycan-laminin interaction in peripheral nerve. We demonstrate that (1) α-dystroglycan is an extracellular peripheral membrane glycoprotein that links β-dystroglycan in the Schwann cell outer membrane with laminin-2 in the endoneurial basal lamina, and (2) dystrophin homologues Dp116 and utrophin are cytoskeletal proteins of the Schwann cell cytoplasm. We also present data that suggest a role for glycosylation of α-dystroglycan in the interaction with laminin.

Notes: Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca2+ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca2+-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µM BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. NG-Monomethyl-l-arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca2+ concentration ([Ca2+]i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca2+]i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca2+]i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca2+ was reduced by pretreating the cells with NaF. The reduction in Ca2+ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca2+]i, and 5-HT2A receptor function in C6 glioma cells.

Notes: Abstract: Myelin vesicles, reconstituted liposomes with proteolipid protein (PLP), the main protein component of myelin, and electrophysiological patch-clamp are potentially powerful tools to study the role of myelin in functional ionic channels. However, technical difficulties in the vesiculation of myelin and the small size of the vesicles obtained do not permit the application of micropipettes for current recordings. From a suspension of purified myelin we have prepared oligolamellar vesicles (mean diameter of 144 nm) using the so-called French pressure system. From this preparation we obtained giant myelin vesicles ∼10 µm in mean diameter, using a dehydration-rehydration procedure. Qualitative analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no significant loss of any component in these vesicles due to pressure, in comparison with non-vesiculated myelin. A way of preparing giant liposomes of ∼80–100 µm and proteoliposomes of ∼30 µm in mean diameter, using the same dehydration-rehydration procedure, is also reported. Reconstitution of purified PLP in giant liposomes was confirmed by fluorescent labeling of PLP and by fluorescence microscopy. The current recordings from these vesicles prove the validity of these methods and provide significant evidence of the existence of ionic channels in myelin membranes and the possibility that PLP functions as a channel. The physiological significance and characterization of these channels remain yet unresolved. These results have a special significance for elucidating the molecular role of myelin in the regulation of neural activity and in the brain ion microenvironment.

Notes: Abstract: We have identified two novel peptide toxins from molluscivorous Conus species that discriminate subtypes of high voltage-activated (HVA) calcium currents in molluscan neurons. The toxins were purified using assays on HVA calcium currents in the caudodorsal cells (CDCs) of the snail Lymnaea stagnalis. The CDC HVA current consists of a rapidly inactivating, transient current that is relatively insensitive to dihydropyridines (DHPs) and a slowly inactivating, DHP-sensitive L-current. The novel toxins, designated ω-conotoxins PnVIA and PnVIB, completely and selectively block the transient HVA current in CDCs with little (PnVIA) or no (PnVIB) effect on the sustained L-type current. The block is rapid and completely reversible. It is noteworthy that both PnVIA and PnVIB reveal very steep dose dependences of the block, which may imply cooperativity in toxin action. The amino acid sequences of PnVIA (GCLEVDYFCGIPFANNGLCCSGNCVFVCTPQ) and of PnVIB (DDDCEPPGNFCGMIKIGPPCCSGWCFFACA) show very little homology to previously described ω-conotoxins, although both toxins share the typical ω-conotoxin cysteine framework but have an unusual high content of hydrophobic residues and net negative charge. These novel ω-conotoxins will facilitate selective analysis of the functions of HVA calcium channels and may enable the rational design of drugs that are selective for relevant subtypes.

Notes: Abstract: This study analyzed the effects of acute systemic treatment with buthionine sulfoximine (BSO), a synthesis inhibitor of the antioxidant reduced glutathione (GSH), on dopaminergic neurons of the murine nigrostriatal pathway. Part 1 of the study established a dose-response curve and the temporal pattern of GSH loss and recovery in the substantia nigra and striatum following acute BSO treatment. Part 2 of the study determined the effect of acute BSO treatment on the morphology and biochemistry of nigrostriatal neurons. We found that decreases in GSH levels had profound morphological effects, including decreased catecholamine fluorescence per cell, increased levels of lipid peroxidation and lipofuscin accumulation, and increased numbers of dystrophic axons in dopaminergic neurons of the nigrostriatal pathway. However, no measurable effects were observed in biochemical levels of either dopamine or its metabolites. These changes mimic those that have been reported to occur in the nigrostriatal system of rodents with advancing age. Our data suggest that reduction of GSH via BSO treatment results in the same types of nigrostriatal degenerative effects that occur during the aging process and consequently is a good model system for examining the role of GSH in protecting this area of the brain against the harmful effects of age-related oxidative stress.

Notes: Abstract: We addressed the balance between thrombin and its serpin protease nexin I (PNI) after sciatic nerve injury in the mouse. Prothrombin levels increased twofold 24 h after nerve crush, as measured by a specific chromogenic assay, and peaked at day 3. Thrombin activity also increased 2–4 days after injury in distal sciatic nerve segments. Nerve RNA analysis using reverse transcriptase-polymerase chain reaction (RT-PCR) assay confirmed that prothrombin was synthesized locally. We also monitored PNI levels in these injured nerve samples by complex formation with an 125I-labeled target protease and found peak activity occurring later, 6–9 days after the thrombin induction. These data indicate that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush-induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. By immunocytochemistry with anti-PNI antibody, we found that activated Schwann cells were the source of induced PNI. These results support the concept that the balance between serine proteases and their serpins is dysregulated during nerve injury and suggests a role for its reestablishment in nerve damage repair.

Notes: Abstract: The release of adenosine and ATP evoked by electrical field stimulation in rat hippocampal slices was investigated with the following two patterns of stimulation: (1) a brief, high-frequency burst stimulation (trains of stimuli at 100 Hz for 50 ms applied every 2 s for 1 min), to mimic a long-term potentiation (LTP) stimulation paradigm, and (2) a more prolonged (3 min) and low-frequency (5 Hz) train stimulation, to mimic a long-term depression (LTD) stimulation paradigm. The release of ATP was greater at a brief, high-frequency burst stimulation, whereas the release of [3H]adenosine was slightly greater at a more prolonged and low-frequency stimulation. To investigate the source of extracellular adenosine, the following two pharmacological tools were used; α,β-methylene ADP (AOPCP), an inhibitor of ecto-5′-nucleotidase, to assess the contribution of the catabolism of released adenine nucleotides as a source of extracellular adenosine, and S-(4-nitrobenzyl)-6-thioinosine (NBTI), an inhibitor of adenosine transporters, to assess the contribution of the release of adenosine, as such, as a source of extracellular adenosine. At low-frequency stimulation, NBTI inhibited by nearly 50% the evoked outflow of [3H]adenosine, whereas AOPCP inhibited [3H]adenosine outflow only marginally. In contrast, at high-frequency stimulation, AOPCP inhibited by 30% the evoked release of [3H]adenosine, whereas NBTI produced a 40% inhibition of [3H]adenosine outflow. At both frequencies, the kinetics of evoked [3H]adenosine outflow was affected in different manners by AOPCP and NBTI; NBTI mainly depressed the rate of evoked [3H]adenosine outflow, whereas AOPCP mainly inhibited the later phase of evoked [3H]adenosine accumulation. These results show that there is a simultaneous, but quantitatively different, release of ATP and adenosine from rat hippocampal slices stimulated at frequencies that can induce plasticity phenomena such as LTP (100 Hz) or LTD (5 Hz). The source of extracellular adenosine is also different according to the frequency of stimulation; i.e., at a brief, high-frequency stimulation there is a greater contribution of released adenine nucleotides for the formation of extracellular adenosine than at a low frequency with a more prolonged stimulation.

Notes: Abstract: A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.

Notes: Abstract: The effects of alcohol and Ca2+ transport inhibitors on depolarization-induced stimulation of oxidative phosphorylation and free-Ca2+ concentrations in rat synaptosomes were investigated. Glucose oxidation was stimulated by depolarization with K+ or veratridine and by the Ca2+ ionophore ionomycin. The stimulation by K+, veratridine, and ionomycin was correlated with elevation of synaptosomal free Ca2+. Depolarization-stimulated respiration was inhibited by verapamil, Cd2+, and ruthenium red but not by diltiazem. Synaptosomal Ca2+ elevation was inhibited by verapamil but not by ruthenium red. These results indicate that the stimulation depends on elevation of mitochondrial free Ca2+. Ethanol, at pharmacological concentrations (50–200 mM), inhibited the Ca2+-dependent stimulation of oxidative phosphorylation. This inhibition resulted, in part, from the inhibition of voltage-gated Ca2+ channels, which inhibited the elevation of synaptosomal free Ca2+, and, in part, from the stimulation of the mitochondrial Ca2+/Na+ antiporter, which inhibited the elevation of the mitochondrial matrix free Ca2+. The inhibition by ethanol of the excitation-induced stimulation of oxidative phosphorylation in the synapse may contribute to the depressant and narcotic effects of alcohol and enhance excitotoxicity.

Notes: Abstract: Previous studies examining age differences in membrane fluidity and cholesterol content have reported on the average or total change in membrane structure, respectively. However, a membrane consists of an exofacial leaflet and a cytofacial leaflet that differ in fluidity and cholesterol distribution. The purpose of the present experiments was to determine fluidity and cholesterol distribution of the exofacial and cytofacial leaflets of brain synaptic plasma membranes (SPMs) from 3–4-, 14–15-, and 24–25-month-old C57BL/6NNIA mice by using trinitrobenzenesulfonic acid (TNBS)-quenching techniques and fluorescent probes. The exofacial leaflet of SPMs from young mice was significantly more fluid compared with the cytofacial leaflet. The large difference in fluidity between the two leaflets was abolished in SPMs of the oldest age group. Total SPM cholesterol and the cholesterol-to-phospholipid molar ratio did not differ among the three different age groups of mice. However, considerable differences were observed in the distribution of cholesterol in the two SPM leaflets. The exofacial leaflet contained substantially less cholesterol than did the cytofacial leaflet (13 vs. 87%, respectively) in SPMs of young mice. This asymmetric distribution of cholesterol was significantly modified with increasing age. There was an approximately twofold increase in exofacial leaflet cholesterol in the oldest group compared with the youngest age group. Transbilayer fluidity and cholesterol asymmetry were altered in SPMs of older mice. This approach is a new and different way of viewing how aging modifies membrane structure. Age differences in SPM leaflet structure may be an important factor regulating activity of certain membrane proteins.

Notes: Abstract: Oocytes from the frog Xenopus laevis were shown recently to express native nicotinic acetylcholine receptors after injection with purified Torpedo electroplaque membrane vesicles. Injection of Xenopus oocytes with rat cortical or nigral synaptosomes has now been shown to result in the expression of γ-aminobutyric acid type A (GABAA) receptor-mediated Cl− currents. Electrophysiological characterization of the responses of these receptors to GABA and other agents revealed that they were incorporated into the oocyte membrane and that they retained their original pharmacological properties, such as sensitivity to Cl− channel blockers, benzodiazepines, and general anesthetics. These results suggest that this approach to the expression of heterologous proteins in Xenopus oocytes may facilitate the study of native synaptic proteins derived from brain tissue.

Notes: Abstract: It has recently been considered that free radicals are closely involved in the pathogenesis of Parkinson's disease (PD), and the level of nitric oxide radical (•NO), one of the free radicals, is reported to increase in PD brain. In the present study, we established a direct detection system for •NO in an in vitro •NO-generating system using 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propanamine as an •NO donor and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO) by electron spin resonance (ESR) spectrometry and examined the quenching effects of the dopamine agonists pergolide and bromocriptine on the amount of •NO generated. •NO appeared to be scavenged by pergolide and, to a lesser extent, by bromocriptine. In the competition assay, the 50% inhibitory concentration values for pergolide and bromocriptine were estimated to be ∼23 and 200 µM, respectively. It was previously reported that in vivo treatment of pergolide and bromocriptine completely protected against the decrease in levels of striatal dopamine and its metabolites in the 6-hydroxydopamine-injected mouse. Considering these findings, pergolide and probably bromocriptine may also protect against dysfunction of dopaminergic neurons because of its multiple effects; not only does it stimulate the presynaptic autoreceptors, but it also directly scavenges •NO radicals and hence protects against •NO-related cytotoxicity. This ESR spectrometry method using carboxy-PTIO may be useful for screening other drugs that can quench •NO.

Notes: Abstract: Myelin/oligodendrocyte glycoprotein (MOG) is a CNS-specific integral membrane protein that is an atypical member of the immunoglobulin (Ig) superfamily with two potential transmembrane domains based upon hydropathy analysis. With only one other exception, all Ig family members possess a single or no membrane spanning region. In order to analyze MOG membrane topology, we prepared stably transfected cells that express mouse MOG and used three domain-specific antisera to ascertain the localization of these hydrophilic domains. As expected, MOG's glycosylated N-terminal Ig-like domain was identified as extracellular, because membrane permeabilization was not required for immunoreactivity with the MOG1–125 antiserum. In contrast, both MOG154–169 and MOG198–218 antisera stained cells only upon permeabilization. These data indicate that only MOG's N-terminal hydrophobic domain spans the lipid bilayer, and we propose that MOG's C-terminal hydrophobic domain associates with the cytoplasmic face of the plasma membrane. As for MOG's second hydrophobic domain, it is clear that either orientation (transmembrane versus membrane-associated) would be unique among Ig-like proteins, and the implications of our proposed topology for MOG in oligodendroglial plasma membrane are discussed.

Notes: Abstract: Significant densities of mRNA for the dopamine D4 receptor in cerebral cortex, particularly in frontal lobe, have been reported in rats and monkeys, supporting the D4 hypothesis in the pathology of schizophrenia. Using northern blot analysis and the competitive reverse transcription-polymerase chain reaction (RT-PCR) method, we determined the relative levels of D4 mRNA in human brain regions to clarify whether the cortical level is also higher in humans. Northern blot analysis revealed an unexpected profile of D4 mRNA in the brain. The detected mRNA size, 1.5 kb, was quite different from the 5.3 kb reported in human neuroblastoma SK-N-MC cells. Higher levels of D4 mRNA were detected not only in the mesolimbic system but also in the corpus callosum, spinal cord, medulla, and subthalamic nucleus. It was surprising that in the cerebral cortex regions as well as the striatum, D4 mRNA was hardly detected. The competitive RT-PCR revealed these relative densities to be at least three orders of magnitude lower than that of the striatal D2 receptor. Our results demonstrate a remarkable difference in cortical D4 mRNA density in humans compared with that in rats and monkeys. Furthermore, the mRNA distribution suggests that the higher density of D4-like binding sites reported recently in normal human striatum is not due to the D4 receptor.

Notes: Abstract: Several lines of evidence indicate that a rapid loss of protein kinase C (PKC) activity may be important in the delayed death of neurons following cerebral ischemia. However, in primary neuronal cultures, cytotoxic levels of glutamate have been reported not to cause a loss in PKC as measured by immunoblot and conventional activity methods. This apparent contradiction has not been adequately addressed. In this study, the effects of cytotoxic levels of glutamate, NMDA, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on membrane PKC activity was determined in cortical neurons using an assay that measures only PKC that is active in isolated membranes, which can be used to differentiate active enzyme from that associated with membranes in an inactive state. A 15-min exposure of day 14–18 cortical neurons to 100 µM glutamate, AMPA, or NMDA caused a rapid and persistent loss in membrane PKC activity, which by 4 h fell to 30–50% of that in control cultures. However, the amount of enzyme present in these membranes remained unchanged during this period despite the loss in enzyme activity. The inactivation of PKC activity was confirmed by the fact that phosphorylation of the MARCKS protein, a PKC-selective substrate, was reduced in intact neurons following transient glutamate treatment. By contrast, activation of metabotropic glutamate receptors by trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid was not neurotoxic and induced a robust and prolonged activation of PKC activity in neurons. PKC inactivation by NMDA and AMPA was dependent on extracellular Ca2+, but less so on Na+, although cell death induced by these agents was dependent on both ions. The loss of PKC activity was likely effected by Ca2+ entry through specific routes because the bulk increase in intracellular free [Ca2+] effected by the Ca2+ ionophore ionomycin did not cause the inactivation of PKC. The results indicate that the pattern of PKC activity in neurons killed by glutamate, NMDA, and AMPA in vitro is consistent with that observed in neurons injured by cerebral ischemia in vivo.

Notes: Abstract: Astrocytes exposed to hypoxia (H) or hypoxia/reoxygenation (H/R) maintain cell viability and display changes in protein biosynthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocytes exposed to H showed induction of an ≈78-kDa polypeptide that demonstrated sequence identity with glucose-regulated protein (GRP) 78. Cell lysates from H/R astrocytes displayed induction of neuroprotective interleukin (IL) 6, which was present in a high-molecular-weight complex also containing GRP78, suggesting that GRP78 might be functioning as a chaperone during cellular stress consequent on H/R. Introduction of anti-sense oligonucleotide to GRP78 into astrocytes prevented expression of the protein and suppressed H/R-induced astrocyte release of IL-6 by ≈50%. These data indicate that modulation of astrocyte properties during oxygen deprivation results, in part, from intracellular glucose depletion and subsequent expression of GRP78, which sustains generation of neuroprotective IL-6 under the stress of H/R.

Notes: Abstract: Mitogen-activated protein (MAP) kinases have been implicated in multiple responses to extracellular stimuli. In this study we show that MAP kinase activity is enhanced after a KCI pulse. This activation correlates with an increased tyrosine phosphorylation of a 42-kDa protein as determined by antiphosphotyrosine immunoblot. The same band is found in an anti-MAP kinase immunoblot. Activity is enhanced within 1 min, reaches a maximum at 2 min, and returns to basal level after 10 min. A second peak of activity is observed between 12 and 30 min. The activation is completely blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), showing the involvement of the AMPA type of glutamate receptor. Partial inhibition of MAP kinase activation by 2-amino-5-phosphonovalerate (APV) also shows the involvement of the NMDA receptor. Because the KCI pulse used induces long-term potentiation (LTP) in rat hippocampal slice, we conclude that MAP kinase may be involved in neuronal transduction events leading to LTP.

Notes: Abstract: Differentiation of NG108-15 neuroblastoma cells following exposure to either 1.5% dimethyl sulfoxide (DMSO)/0.5% fetal bovine serum (FBS) or serum starvation resulted in significant differences in angiotensin (AT) receptor levels and the AT1/AT2 receptor ratio. When NG108 cells were differentiated for 4 days with DMSO/low serum, the number of AT binding sites increased 30-fold compared with the binding levels on undifferentiated (blast) cells. However, cells differentiated by serum starvation for 4 or 14 days resulted in only a modest 2.5- and fivefold increase in AT receptor levels, respectively, over the levels seen with the undifferentiated cells. KD values for all treatment conditions were not significantly different (0.71 ± 0.11 nM, p = 0.06). Using the AT1 and AT2 isoform-specific receptor antagonists losartan and PD123319, the relative numbers of AT receptor subtypes on undifferentiated and differentiated cells were determined by competitive inhibition against 125I-[Sar1,Ile8]-angiotensin II (sarile). A majority of the AT receptors on undifferentiated NG108 cells were the AT1 subtype (AT1/AT2 receptor ratio of 8:3). Differentiation by serum starvation and DMSO/low serum treatment resulted in fivefold and 30-fold increases in AT receptor levels, respectively, compared with the levels seen with the undifferentiated cells. Although serum starvation increased the total number of AT1 and AT2 receptors, it did not significantly alter the AT1/AT2 receptor ratio. In contrast, differentiation with DMSO/low serum both increased the total number of AT1 and AT2 receptors and reversed the AT1/AT2 receptor ratio (1:3). The increase in AT receptors following differentiation with DMSO/low serum for 4 days was largely accounted for by an 80-fold increase in the AT2 receptor level. Previous studies by Tallant et al. (1991) and Bryson et al. (1992) reported increased AT2 receptor expression following neuroblastoma differentiation with dibutyryl cyclic AMP and DMSO/low serum, respectively, and suggested a role for the AT2 receptor in neuronal differentiation. In the present study, we have extended these earlier observations by demonstrating that the method of differentiation significantly affects both the AT receptor level and the ratio of AT1 to AT2 receptor expression. Finally, our findings indicate that the AT2 receptor is expressed as a consequence of neuronal maturation and does not mediate morphological differentiation.

Notes: Abstract: Imprinting in chicks is a form of juvenile learning that has been used to study the basic cellular mechanisms of learning and memory. The forebrain area mediorostral neostriatum/hyperstriatum ventrale (MNH) is a center for acoustic imprinting. Electrophysiological and pharmacological behavioral studies in the MNH have demonstrated that the glutamatergic system and the associated receptors are critically involved in auditory filial imprinting. Accordingly, we investigated the hypothesis that stimulus-evoked glutamate release may be altered after this learning process. Using an in vivo microdialysis technique, we observed a significantly higher increase of extracellular glutamate level in tone-imprinted chicks during exposure to the previously imprinted tone than in socially imprinted control chicks. In a further series of experiments, where we exposed animals from both experimental groups to handling distress, glutamate levels in MNH showed only a slight increase, whereas we observed a pronounced increase of extracellular glutamate in the lobus parolfactorius (LPO), the avian analogue of the basal ganglia. No difference of distress-evoked glutamate release was found in MNH and LPO between tone-imprinted and socially imprinted chicks. The tone-evoked enhanced glutamate response in tone-imprinted chicks suggests that during auditory imprinting glutamatergic synapses develop the potential to increase transmitter release in response to the imprinting stimulus.

Notes: Abstract: Thioperamide (2 mg/kg, i.p.), a histamine H3-receptor antagonist, increased the number of somatostatin (SS) receptors, with no change in the affinity constant, in the rat frontoparietal cortex. This effect was prevented by treatment with (R)-α-methylhistamine (3.2 mg/kg, i.p.), a histamine H3-receptor agonist. Thioperamide also induced an increase in SS binding in rats pretreated with mepyramine, a histamine H1-receptor antagonist, or cimetidine, a histamine H2-receptor antagonist. Pretreatment with mepyramine plus cimetidine administered simultaneously antagonized the thioperamide effect on SS binding. The increase in the number of SS receptors was accompanied by a greater SS-mediated inhibition of basal and forskolin-stimulated adenylyl cyclase (AC) activity in frontoparietal cortical membranes in the thioperamide group. Furthermore, the functional activity of the guanine nucleotide-binding inhibitory protein (Gi protein) was not altered by thioperamide or (R)-α-methylhistamine administration in frontoparietal cortical membranes. In rats treated with mepyramine plus thioperamide or cimetidine plus thioperamide, the increase in the number of SS receptors was also accompanied by an increased SS inhibition of AC activity. Thioperamide induced a significant increase in SS-like immunoreactivity content in the frontoparietal cortex. Altogether, these results suggest that frontoparietal cortical histamine may play, at least in part, a role in the regulation of the somatostatinergic system.

Notes: Abstract: Using acutely isolated rat substantia nigra neurons, our previous studies indicated that sulfated cholecystokinin octapeptide (CCK-8) excites substantia nigra dopaminergic neurons by increasing the cationic conductance and that pertussis toxin-insensitive G proteins mediate CCK-8 induction of cationic currents. Gαq and Gα11 are expressed in various tissues, including the brain, and likely to mediate pertussis toxin-insensitive neural signal transductions. In the present study, two different experiments were performed to test the hypothesis that Gαq/11 mediates CCK-8 enhancement of the cationic conductance. First, we investigated the expression of Gαq and Gα11 mRNAs in CCK-8-responsive substantia nigra dopaminergic neurons by combining whole-cell patch-clamp recordings with a single-cell reverse transcriptase-polymerase chain reaction assay. After CCK-8-evoked cationic currents were recorded, cellular RNA was harvested from single neurons and used as a template for the subsequent reverse transcriptase-polymerase chain reaction analysis. Gαq and Gα11 mRNAs were present in all substantia nigra dopaminergic neurons that responded to CCK-8. Substantia nigra dopaminergic neurons were also internally perfused with the antibody raised against the common C-terminus of Gαq and Gα11 during whole-cell recordings. CCK-8 failed to induce cationic currents after dopaminergic neurons were dialyzed with the anti-Gαq/11 antibody. Our studies suggest that CCK-8 activation of the cationic conductance in substantia nigra dopaminergic neurons is transduced by Gαq and/or Gα11.

Notes: Abstract: The contribution of NMDA receptors to regulation of serotonin (5-HT) release was assessed by in vivo microdialysis in freely behaving rats. During infusion of NMDA (30, 100, and 300 µM) into the dorsal raphe nucleus (DRN), 5-HT was increased by ∼25, 100, and 280%, respectively. Competitive and noncompetitive NMDA-receptor antagonists blocked this effect on DRN 5-HT. Infusion of NMDA (300 µM) into the DRN also produced an 80% increase in extracellular 5-HT in the nucleus accumbens. During infusion of NMDA (100 and 300 µM) into the median raphe nucleus (MRN), 5-HT was increased by ∼15 and 80%, respectively. NMDA-receptor antagonists blocked this effect on MRN 5-HT. Infusion of NMDA into the MRN also produced a significant increase in hippocampal 5-HT. In contrast, infusion of NMDA into the nucleus accumbens, frontal cortex, or hippocampus produced small decreases in 5-HT in these forebrain sites. Taken together, these results suggest that NMDA receptors in the midbrain raphe, but not the forebrain, can have an excitatory influence on 5-HT neurons and, thus, produce increased 5-HT release in the forebrain. Furthermore, in comparison with the MRN, DRN 5-HT neurons were more sensitive to the excitatory effect of NMDA.

Notes: Abstract: The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian α, β, γ, and δ isozymes show the greatest divergence from each other. To investigate the function of this variable region, we have characterized the catalytic and structural properties of six of the Drosophila isoforms. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologues of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences were observed. First, the R3A isoform was found to have a significantly higher Kact for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted was that the R6 isoform had a significantly lower Km for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity.

Notes: Abstract: Secretoneurin is a 33-amino-acid peptide produced in vivo from secretogranin II. An antiserum raised against this peptide recognizes both the free peptide and its precursors. By HPLC and radioimmunoassay we characterized the immunoreactive molecules and determined the levels of immunoreactivity in various rat organs. In adrenal medulla and to a lesser degree in the anterior pituitary processing of secretogranin II to secretoneurin was very limited, whereas in all other organs studied (brain, intestine, endocrine pancreas, thyroid gland, and posterior pituitary) a high degree of processing was apparent. Thus, practically all of the immunoreactivity was present as free secretoneurin. This was also true for serum. When the total amount of secretoneurin immunoreactivity was calculated for the various organs, the largest pools in descending order were in the intestine, CNS, anterior pituitary, pancreas, and adrenal gland. This makes it likely that secretoneurin in serum is mainly derived from the intestine. The high degree of processing of secretogranin II in most organs is consistent with the concept that this protein acts as a precursor of a functional peptide, i.e., secretoneurin.

Notes: Abstract: Somatostatin (SS) is a neuropeptide that is distributed in various regions of the CNS, where it may act as a neurotransmitter or neuromodulator. SS produces multiple effects in the CNS through interactions with membrane receptors. In particular, SS inhibits various secretory responses in different cell types. In the present study, we have investigated the effects of exogenous application of SS on the intracellular free Ca2+ concentration ([Ca2+]i) in PC12 cells, a rat pheochromocytoma cell line. SS did reduce the magnitude of the secondary, maintained Ca2+ influx brought about by K+ depolarization. Similar effects were obtained with the application of SS analogues, such as d-Trp8-SS, d-Trp8-d-Cys14-SS, CGP-23996, and SMS-201995. In addition, treatment with cyclo-SS, a SS antagonist, did not alter [Ca2+]i. Experiments with selective blockers of different voltage-dependent Ca2+ channels, such as methoxyverapamil (D600) and Ω-conotoxin GVIA, demonstrated that the effects of SS on [Ca2+]i were mediated by voltage-dependent Ca2+ channels of the L type. Control experiments with a membrane potential indicator, i.e., the fluorescent dye bisoxonol, excluded that SS influenced the level of the membrane potential. SS effects on PC12 cells suggest the possibility that this neuropeptide plays a role in the modulation of cell functional activity by altering Ca2+ influx.

Notes: Abstract: We have previously reported that insulin/insulin-like growth factor (IGF)-I induced the α1 isoform of Na+,K+-ATPase in cultured astrocytes. In this study the effects of insulin/IGF-I on Na+,K+-ATPase activity and cell proliferation were examined in astrocytes cultured under the various conditions, to test the possible involvement of the enzyme activity in the mitogenic action of IGF-I on astrocytes. Insulin increased Na+,K+-ATPase activity and stimulated cell proliferation in subconfluent astrocytes (cultured for 7–14 days in vitro). In contrast, these effects were not observed in confluent cells (cultured for 28 days). Furthermore, insulin stimulated neither the enzyme activity nor [3H]thymidine incorporation in astrocytes preincubated in fetal calf serum-free medium for 2 days (quiescent cells) and treated with dibutyryl cyclic AMP (differentiated cells). The increases in Na+,K+-ATPase activity and expression of the α1 mRNA preceded the mitogenic effect. 125I-IGF-I binding experiment showed that all the cells used here had similar binding characteristics. The insulin-induced increase in enzyme activity was not affected by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), and it was observed even in Ca2+-free medium. The stimulation by IGF-I of [3H]thymidine incorporation was attenuated by ouabain and a low external K+ level. These findings suggest that stimulation of Na+,K+-ATPase activity is involved in the mitogenic action of IGF-I on cultured astrocytes.

Notes: Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 mM K+. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10−5M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.

Notes: Abstract: Phosphatidylinositol 4,5-bisphosphate (PIP2) is absolutely required for the ADP-ribosylation factor-stimulated phospholipase D (PLD) activity. In the present study, partially purified rat brain PLD was found to be activated by another PLD activator, RhoA, when PIP2, but not other acidic phospholipids, was included in vesicles comprising phosphatidylethanolamine (PE) and the PLD substrate phosphatidylcholine (PC) (PE/PC vesicles), demonstrating the absolute requirement of PIP2 for the RhoA-stimulated PLD activation, too. It is interesting that the RhoA-dependent PLD activity in the partially purified preparation was drastically decreased after the preparation was incubated with and separated from PE/PC vesicles containing PIP2. The PLD activity was extracted by higher concentrations of NaCl from the vesicles containing PIP2 that were incubated with and then separated from the partially purified PLD preparation. These results demonstrate that RhoA-dependent PLD binds to PE/PC vesicles with PIP2. The degree of binding of the RhoA-dependent PLD activity to the vesicles was totally dependent on the amount of PIP2 in the vesicles and correlated well with the extent of the enzyme activation. Furthermore, it was found that a recombinant peptide of the pleckstrin homology domain of β-adrenergic receptor kinase fused to glutathione S-transferase, which specifically binds to PIP2, inhibited the PIP2-stimulated, RhoA-dependent PLD activity in a concentration-dependent manner. From these results, it is concluded that in vitro rat brain PLD translocates to the vesicles containing PIP2, owing to its specific interaction with PIP2, to access its substrate PC, thereby catalyzing the hydrolysis of PC. PLD appears to localize exclusively on plasma membranes of cells and tissues. An aminoglycoside, neomycin, that has high affinity for PIP2 effectively extracted the RhoA-dependent PLD activity from rat brain membranes. This indicates that PIP2 serves as an anchor to localize PLD on plasma membranes in vivo.

Notes: Abstract: We investigated transcriptional regulation of the rat tyrosine hydroxylase (TH) gene by muscarinic stimulation in human neuroblastoma SK-N-BE(2)M17 cells. Carbachol treatment increased the levels of intracellular Ca2+ and inositol 1,4,5-trisphosphate (IP3) and enhanced transcription of the TH gene. The muscarinic receptor antagonist atropine completely abolished the carbachol effect on TH gene expression. When cells were loaded with 50 µM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) to chelate intracellular Ca2+, carbachol still raised intracellular IP3 level and enhanced TH gene expression. Transient transfection analysis of the 5′ upstream region of TH gene revealed that the AP1 cis-acting element at −205 to −199 bp was responsible for carbachol stimulation. But carbachol did not enhance TH gene expression in protein kinase C (PKC)-activated or down-regulated cells that had been induced by 5-min or 24-h exposure to phorbol 12-myristate 13-acetate (PMA), respectively. Thus, Ca2+-independent PKC may play a role in carbachol-induced TH gene expression. We demonstrated by gel retardation and competition assays that a DNA sequence containing the wild-type AP1 site formed the specific DNA-protein complex. However, treatment with carbachol or PMA did not change the amount of the specific DNA-protein complex. Our results indicate that stimulation of phospholipase C-linked muscarinic receptors leads to elevated TH gene expression via AP1-mediated enhancement in a PKC-dependent pathway.

Notes: Abstract: Ser55 within the head domain of neurofilament light chain (NF-L) is transiently phosphorylated by protein kinase A, and phosphorylation of this residue is thought to regulate assembly of neurofilaments. To understand how Ser55 phosphorylation influences NF-L assembly, wild-type and mutant NF-L genes in which Ser55 was mutated to alanine, so as to prevent phosphorylation, or to aspartate, so as to mimic permanent phosphorylation, were transfected into mammalian cells that contain or do not contain an endogenous intermediate filament network. Wild-type and mutant NF-Ls localised to the Triton X-100-insoluble fraction, which suggests that phosphorylation of Ser55 does not inhibit assembly of NF-L and NF-L/vimentin polymers at or below the tetrameric stage. Immunofluorescence microscopy of transfected cells demonstrated that the wild-type and mutant NF-Ls all colocalised with vimentin to produce similar filamentous arrays. However, in cells lacking an endogenous intermediate filament network, the aspartate mutant produced a pattern of staining different from that of the wild-type or alanine mutant. These results suggest that phosphorylation of NF-L Ser55 is not a mechanism that precludes assembly of neurofilaments from monomers into intermediate filament structures but that phosphorylation/dephosphorylation of this residue might confer more subtle characteristics on neurofilament assembly properties and architecture.

Notes: Abstract: Northern blot analysis determined whether multiple α4 transcripts for neuronal nicotinic receptors in rat brain could be detected as distinct bands. When poly(A)+ RNA was isolated from brain regions and hybridized with a Hinfl fragment of α4-1 cDNA containing a sequence shared by both α4-1 and α4-2, but little homology with other α or β subunits, bands at 6.0, 4.6, and 2.6 kb were obtained. When a Taql fragment with selectivity for α4-1 was used, a single band was present at 6.0 kb. The 6.0-kb band was least abundant in all brain regions; the 2.6-kb band was most abundant in frontal cortex, hippocampus, striatum, basal forebrain, and thalamus, whereas the 4.6-kb band was most abundant in midbrain and cerebellum. Nicotine (3.6 µmol/kg, s.c., twice daily) increased the abundance of the 4.6-kb transcript in frontal cortex significantly by 28% following 2.5 days of injections; the 6.0- and 2.6-kb transcripts were unchanged. Nicotine did not affect α4 transcripts in other brain regions. Results suggest that increased mRNA levels may mediate the nicotine-induced up-regulation of receptors in cerebral cortex.

Notes: Abstract: Astrocytes have been shown to express endothelin (ET) receptors functionally coupled, via different heterotrimeric G proteins, to several intracellular pathways. To assess the relative contribution of each subtype in the astrocytic responses to ET-1, effects of BQ123, an antagonist selective for the ET receptor subtype A (ETA-R), and IRL1620, an agonist selective for the ET receptor subtype B (ETB-R), were investigated in primary cultures of rat astrocytes. Binding experiments indicated that the ETB-R is the predominant subtype in these cells. Inhibition of forskolin-stimulated cyclic AMP production was observed under ETB-R stimulation. Bordetella pertussis toxin (PTX) pretreatment completely abolished this effect, indicating that this pathway is coupled to the ETB-R via Gi protein. Increases of tyrosine phosphorylation of cellular proteins, stimulation of mitogen-activated protein kinase (MAPK), and DNA synthesis were also found to be mediated by the ETB-R, but through PTX-insensitive G protein. IRL1620-induced MAPK activation involved the adapter proteins Shc and Grb2 and the serine/threonine kinase Raf-1. This study reveals that the various effects of ET-1 in astrocytes are mediated by the ETB-R, which couples to multiple signaling pathways including the MAPK cascade.

Notes: Abstract: Incubation with l-DOPA induced a rise in GSH level in cultures of fetal rat mesencephalon, mouse neuroblastoma (Neuro-2A), human neuroblastoma (SK-N-MC), pig kidney epithelial cells (LLC-PK1), and glia from newborn rat brain, but not C6 glioma cells or neuronal cultures (no glia) from the mesencephalon. The pure neuronal cultures were destroyed by incubation with l-DOPA; added ascorbic acid or superoxide dismutase protected the cells. Washout of l-DOPA after 48 h amplified the rise in GSH content in mixed cultures (neurons plus glia). Examination of structure-activity relationships for elevating GSH levels in responsive cell types revealed that autooxidizable compounds (α-methyl-DOPA, dopamine, apomorphine, catechol, and hydroquinone) behaved similarly to l-DOPA, whereas structural analogues that cannot undergo autooxidation (3-O-methyl-DOPA, tyrosine, 2,4-dihydroxyphenylalanine, and resorcinol) failed to elevate GSH levels. Therefore, up-regulation of GSH appears to be a response to a mild oxidative stress. When mixed mesencephalic cultures were exposed to a strong oxidant stress by incubation with tert-butyl hydroperoxide, a loss in viability was seen. Cultures pretreated with l-DOPA or hydroquinone were protected from loss of viability. However, when cultures were pretreated with both l-DOPA and ascorbate, which prevents the rise in GSH level, protection was lost, in accord with the failure to up-regulate GSH. These results show that the up-regulation of cellular GSH evoked by autooxidizable agents is associated with significant protection of cells. Glia play an essential role in the response of mesencephalic cell cultures. An ability to up-regulate GSH may serve a protective role in vivo.

Notes: Abstract: The endogenous phospholipid mediator lysophosphatidic acid (LPA) caused growth cone collapse, neurite retraction, and cell flattening in differentiated PC12 cells. Neurite retraction was blocked by cytochalasin B and ADP-ribosylation of the small-molecular-weight G protein Rho by the Clostridium botulinum C-3 toxin. LPA induced a transient rise in the level of inositol 1,4,5-trisphosphate, and retraction was blocked by inhibitors of phospholipase β. Repeated application of LPA elicited homologous desensitization of the Ca2+ mobilization response. The activation of the phosphoinositide (PIP)-Ca2+ second messenger system played a permissive role in the morphoregulatory response. Blockers of protein kinase C—chelerythrine, a myristoylated pseudosubstrate peptide, staurosporine, and depletion of protein kinase C from the cells by long-term phorbol ester treatment—all diminished neurite retraction by interfering with LPA-induced Ca2+ mobilization, which was required for the withdrawal of neurites. A brief 15-min treatment with 4β-phorbol 12-myristate 13-acetate also blocked retraction and Ca2+ mobilization, by inactivating the LPA receptor. Inhibition of protein tyrosine phosphorylation by herbimycin diminished retraction. Although activation of the PIP-Ca2+ second messenger system appears necessary for the Rho-mediated rearrangements of the actin cytoskeleton, bradykinin, which activates similar signaling events, failed to cause retraction, indicating that a yet unidentified novel mechanism is also involved in the LPA-induced morphoregulatory response.

Notes: Abstract: Rapid chronoamperometric recordings, using Nafion-coated carbon-fiber electrodes (30–90 µm o.d.), were used to investigate overflow and uptake of dopamine (DA) in the striatum of normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated rhesus monkeys. The monkeys were anesthetized with isoflurane and placed in a stereotaxic apparatus. Magnetic resonance imaging-guided sterile stereotaxic procedures were used for implantations of the electrochemical electrodes coupled with single-barrel micropipettes that were used to apply potassium or DA locally. Potassium evoked a robust overflow of DA-like electrochemical signals into the brain extracellular space in the unlesioned or normal putamen and caudate nucleus of the rhesus monkeys. In contrast, potassium did not produce any detectable changes (〉 97% depletion) of DA in the MPTP-lesioned striatum. In addition, the diffusion/clearance of locally applied DA was markedly altered in the lesioned caudate nucleus and putamen compared with unlesioned striatum. Cell counts of the number of residual tyrosine hydroxylase-positive neurons in MPTP-treated monkeys, in conjunction with whole-tissue levels of DA and its metabolites, showed that the MPTP lesions produced extensive damage of the nigrostriatal DA system. These data indicate that residual dopaminergic fibers remaining after MPTP lesions are dysfunctional and have a greatly diminished capacity for high-affinity DA uptake.

Notes: Abstract: The insulin-like growth factors (IGFs) are trophic factors whose growth-promoting actions are mediated via the IGF-I receptor and modulated by six IGF binding proteins (IGFBPs). In this study, we observed increased transcripts of both IGF-I and IGF-I receptor after rat sciatic nerve transection. Schwann cells (SCs) were the main source of IGF-I and IGFBP-5 immunoreactivity until 7 days after nerve transection, when invading macrophages in the distal nerve stumps were strongly IGF-I positive. In vitro, IGF-I promoted SC mitogenesis. Northern analysis revealed that SCs expressed IGF-I receptor and IGFBP-5. IGF-I treatment increased the intensity of IGFBP-5 without affecting gene expression. Des(1–3)IGF-I, an IGF-I analogue with low affinity for IGFBP, had no such effect. Incubation of recombinant human IGFBP-5 with SC conditioned media revealed IGF-I protection of IGFBP-5 from proteolysis, implying the presence of an IGFBP-5 protease in SC conditioned media. Collectively, these data support the concept that, in response to nerve injury, invading macrophages produce IGF-I and SC express the IGF-I receptor, to facilitate regeneration. This regenerative process may be augmented further by the ability of SC to secrete IGFBPs, which in turn may increase local IGF-I bioavailability.

Notes: Abstract: In the current study we examined the effects of coadministration of a serotonin 5-HT1A antagonist, (±)-1-(1H-indol-4-yloxy)-3-(cyclohexylamino)-2-propanol maleate (LY 206130), and a dual 5-HT and norepinephrine (NE) uptake inhibitor, duloxetine, on extracellular levels of NE, 5-HT, dopamine (DA), 5-hydroxyindoleacetic acid, and 3,4-dihydroxyphenylacetic acid in rat hypothalamus microdialysates. LY 206130 (3.0 mg/kg, s.c.) alone significantly increased NE and DA levels by 60 and 34%, respectively, without affecting 5-HT levels. Duloxetine administration at 4.0 mg/kg, i.p. alone produced no significant changes in levels of 5-HT, NE, or DA. In contrast, when LY 206130 and duloxetine were coadministered at 3.0 mg/kg, s.c. and 4.0 mg/kg, i.p., respectively, 5-HT, NE, and DA levels increased to 5.7-, 4.8-, and threefold over their respective basal levels. These data demonstrate that antagonism of somatodendritic 5-HT1A autoreceptors and concomitant inhibition of 5-HT and NE uptake with duloxetine may promote synergistic increases in levels of extracellular 5-HT, NE, and DA in hypothalamus of conscious, freely moving rats.

Notes: Abstract: The catecholamine precursor l-3,4-dihydroxyphenylalanine (l-DOPA) is used to augment striatal dopamine (DA), although its mechanism of altering neurotransmission is not well understood. We observed the effects of l-DOPA on catecholamine release in ventral midbrain neuron and PC12 pheochromocytoma cell line cultures. In ventral midbrain neuron cultures exposed to 40 mM potassium-containing media, l-DOPA (100 µM for 1 h) increased DA release by 〉10-fold. The elevated extracellular DA levels were not significantly blocked by the DA/norepinephrine transport inhibitor nomifensine, demonstrating that reverse transport through catecholamine-uptake carriers plays little role in this release. In PC12 cells, where DA release from individual secretory vesicles can be observed, l-DOPA (50 µM for 1 h) elevated DA release in high-potassium media by 370%. Amperometric measurements demonstrated that l-DOPA (50 µM for 40–70 min) did not raise the frequency of vesicular exocytosis but increased the average size of quantal release to at least 250% of control levels. Together, these findings suggest that l-DOPA can increase stimulation-dependent transmitter release from DA cells by augmenting cytosolic neurotransmitter, leading to increased quantal size.

Notes: Abstract: Heme oxygenase is an essential enzyme in the heme catabolism that produces carbon monoxide (CO). This study was designed to examine the expression of two heme oxygenase isozyme mRNAs in the human brain and to explore the involvement of nitric oxide (NO) and various neuropeptides in the regulation of their expression. Northern blot analysis showed the expression of heme oxygenase-1 and heme oxygenase-2 mRNAs in every region of the brain examined, with the highest levels found in the frontal cortex, temporal cortex, occipital cortex, and hypothalamus. In a human glioblastoma cell line, T98G, treatment with any of three types of NO donors—sodium nitroprusside, 3-morpholinosydnonimine, and S-nitroso-l-glutathione—caused a significant increase in the levels of heme oxygenase-1 mRNA but not in the levels of heme oxygenase-2 and heat-shock protein 70 mRNAs. Sodium nitroprusside increased the levels of heme oxygenase-1 protein but not the levels of heat-shock protein 70 in T98G cells. The increase in content of heme oxygenase-1 mRNA caused by sodium nitroprusside was completely abolished by the treatment with actinomycin D. On the other hand, the levels of heme oxygenase isozyme mRNAs were not noticeably changed in T98G cells following the treatment with 8-bromo cyclic GMP, sodium nitrite, or various neuropeptides, such as calcitonin gene-related peptide, endothelin-1, and corticotropin-releasing hormone. The present study has shown the expression profiles of heme oxygenase-1 and -2 mRNAs in the human brain and the induction of heme oxygenase-1 mRNA caused by NO donors in T98G cells. These findings raise a possibility that the CO/heme oxygenase system may function in concert with the NO/NO synthase system in the brain.

Notes: Abstract: C6 glioma cells were used as a model system to study the regulation of EAAC1-mediated Na+-dependent l-[3H]glutamate transport. Although a 30-min preincubation with forskolin had no effect on transport activity, preincubation with phorbol 12-myristate 13-acetate (PMA) increased transport activity two- to threefold. PMA caused a time-dependent and concentration-dependent increase in EAAC1-mediated l-[3H]glutamate transport activity. A 2-min preincubation with PMA was sufficient to cause more than a twofold increase in transport activity and the protein synthesis inhibitor cycloheximide had no effect on the increase. These data suggest that this increase is independent of protein synthesis. The EC50 value of PMA for stimulation of transport activity was 80 nM. Kinetic analyses demonstrated that the increase in transport activity was due to a 2.5-fold increase in Vmax with no change in Km. PMA also increased the transport of the nonmetabolizable analogue, d-[3H]aspartate to the same extent. In parallel assays, PMA did not, however, increase Na+-dependent glycine transport activity in C6 glioma. The inactive phorbol ester 4α-phorbol 12,13-didecanoate, did not stimulate l-[3H]glutamate transport activity, and the protein kinase C inhibitor chelerythrine blocked the stimulation caused by PMA. Okadaic acid and cyclosporin A, which are phosphatase inhibitors, had no effect on the stimulation of transport activity caused by PMA. The Ca2+ ionophore A23187 did not act synergistically to increase PMA stimulation. In previous studies, PMA caused a rapid increase in amiloride-sensitive Na+/H+ transport activity in C6 glioma. In the present study, pre- and coincubation with amiloride had no effect on the stimulation of transport activity caused by PMA. These studies suggest that activation of protein kinase C causes a rapid increase in EAAC1-mediated transport activity. This rapid increase in Na+-dependent l-[3H]-glutamate transport activity may provide a novel mechanism for protection against acute insults to the CNS.

Notes: Abstract: To study the level of ciliary neurotrophic factor (CNTF) in human nervous tissues, we developed a sensitive enzyme-linked immunoassay using a specific antibody against human CNTF. This method allowed us to detect as little as 0.3 ng/ml of human CNTF with good linearity and accuracy. Using this method, CNTF levels were determined in human sciatic nerves obtained at autopsy from 21 amyotrophic lateral sclerosis (ALS) patients and 48 subjects who had died of other neurological diseases. CNTF genotypes were also determined. The results indicated that CNTF levels were high in the normal homozygotes and approximately halved in the heterozygote subjects. There was, however, no significant difference in CNTF levels in the sciatic nerves between ALS and other neurological disease patients, indicating that the CNTF level was mainly determined by its genotypes and that the level in the sciatic nerves was not reduced in ALS patients.

Notes: Abstract: We observed that retinoic acid, which differentiates the human neuroblastoma SK-N-BE into mature neurons, induced an elevation in levels of polyunsaturated fatty acids, especially arachidonic acid (20:4 n-6). This effect was not induced by phorbol myristate acetate, another differentiating agent. We then explored the effects of retinoic acid on the formation of arachidonic acid and of docosahexaenoic acid from precursors and on the de novo lipid synthesis from acetate at various stages of differentiation, which was assessed by morphological (cell number and neurite outgrowth) and biochemical (protein content and thymidine incorporation) criteria. At 3 days of incubation with retinoic acid, in the n-6 series, total conversion of linoleic acid, especially to 20:3 n-6, was elevated, in association with preferential incorporation of acetate into phospholipids; in contrast, at 8 days, synthesis of 20-carbon polyunsaturated fatty acids declined, in association with enhanced incorporation in triglycerides. In the n-3 series, eicosapentaenoic acid was converted to docosahexaenoic acid in SK-N-BE, but the conversion was not affected by retinoic acid. During the early stage of neuronal differentiation, therefore, enhanced production of 20-carbon polyunsaturated fatty acids from their precursors occurred, and newly formed fatty acids were preferentially incorporated in phospholipids, possibly in association with membrane deposition. When differentiation was completed, arachidonic acid formation and incorporation of acetate in phospholipids and cholesterol declined with enhanced labeling of storage lipids.

Notes: Abstract: Dopamine can oxidize to form reactive oxygen species and quinones, and we have previously shown that dopamine quinones bind covalently to cysteinyl residues on striatal proteins. The dopamine transporter is one of the proteins at risk for this modification, because it has a high affinity for dopamine and contains several cysteinyl residues. Therefore, we tested whether dopamine transport in rat striatal synaptosomes could be affected by generators of reactive oxygen species, including dopamine. Uptake of [3H]dopamine (250 nM) was inhibited by ascorbate (0.85 mM; −44%), and this inhibition was prevented by the iron chelator diethylenetriaminepentaacetic acid (1 mM), suggesting that ascorbate was acting as a prooxidant in the presence of iron. Preincubation with xanthine (500 µM) and xanthine oxidase (50 mU/ml) also reduced [3H]dopamine uptake (−76%). Preincubation with dopamine (100 µM) caused a 60% inhibition of subsequent [3H]dopamine uptake. This dopamine-induced inhibition was attenuated by diethylenetriaminepentaacetic acid (1 mM), which can prevent iron-catalyzed oxidation of dopamine during the preincubation, but was unaffected by the monoamine oxidase inhibitor pargyline (10 µM). None of these incubations caused a loss of membrane integrity as indicated by lactate dehydrogenase release. These findings suggest that reactive oxygen species and possibly dopamine quinones can modify dopamine transport function.

Notes: Abstract: The effect of melatonin on [3H]glutamate uptake and release in the golden hamster retina was studied. In retinas excised in the middle of the dark phase, i.e., at 2400 h, melatonin (0.1 and 10 nM) significantly increased [3H]glutamate uptake, and this effect persisted in a Ca2+-free medium. On the other hand, melatonin significantly increased [3H]glutamate release in retinas excised at 2400 h, but this effect was Ca2+ sensitive. Melatonin significantly increased 45Ca2+ uptake by a crude synaptosomal fraction from retinas of hamsters killed at 2400 h. In retinas excised at 1200 h, melatonin had no effect on [3H]glutamate uptake, [3H]glutamate release, or 45Ca2+ uptake at any concentration tested. Cyclic GMP analogues, i.e., 8-bromoguanosine 3′,5′-cyclic monophosphate and 2′-O-dibutyrylguanosine 3′,5′-cyclic monophosphate, significantly increased [3H]glutamate uptake, [3H]glutamate release, and 45Ca2+ uptake by tissue removed at 1200 and 2400 h, suggesting that the effects of melatonin could correlate with a previously described effect of melatonin on cyclic GMP levels in the golden hamster retina. Taking into account the key role of glutamate in visual mechanisms, the results suggest the participation of melatonin in retinal physiology.

Notes: Abstract: The contribution of N-linked carbohydrates to the function of the human norepinephrine transporter (NET) was investigated using site-directed mutagenesis to inactivate the two most carboxy-terminal (NQQ mutant) or all three (QQQ mutant) sites for N-glycosylation within the extracellular loop between transmembrane domains 3 and 4. In HeLa cells transiently expressing the NET, two glycosylated forms of the transporter at 90 and 60 kDa are immunoprecipitated by NET antisera. A single 50-kDa species is observed in cells expressing the QQQ mutant, and it likely represents the NET core protein. Analyses of substrate transport kinetics showed rank order Vmax of 19:9:1 for NET/NQQ/QQQ without a change in the apparent affinity of the wild-type and mutated carriers for either substrates or transport inhibitors. Cell surface biotinylation indicates that all NET, NQQ, and QQQ transporter species are detected at the plasma membrane but that glycosylated forms are selectively enriched. The transport activities exhibited by each of the carriers correlate well with cell surface content. Subcellular localization of transporters using immunofluorescence microscopy shows that reductions in surface expression and transport are associated with a corresponding increase in the intracellular retention of mutated carriers. Thus, N-linked glycosylation does not alter the apparent affinity of NET for either substrates or inhibitors of transport but, instead, appears to influence the abundance of carriers at the cell surface.

Notes: Abstract: In this study, we have investigated the effect of mivazerol, [3-(1H-imidazol-4-yl)methyl-1]-2-hydroxy-benzamide hydrochloride, a new α2-agonist lacking hypotensive properties and a potential anti-ischemic drug, on the evoked release of norepinephrine, aspartate, and glutamate in tissue preparations from hippocampus, spinal cord T1–T5 section, rostrolateral ventricular medulla, and nucleus tractus solitarii of the brainstem of rat. A simple and efficient in vitro procedure to study pharmacologically the release of norepinephrine and glutamate is described. Tissues were chopped into (0.3 × 0.2 × 0.2 mm3) sections and the resulting minces were used for this study. Exposure to KCl (10–75 mM) for 5 min served as a stimulus for the release response. One, S (for aspartate and for glutamate release), or two such stimuli, S1 and S2 (for norepinephrine release) were conducted. The release of norepinephrine (+150% above baseline) was inhibited in a dose-dependent manner by mivazerol in hippocampus (IC50 = 1.5 × 10−8M), spinal cord (IC50 = 5 × 10−8M), rostrolateral ventricular medulla (IC50 = 10−7M), and nucleus tractus solitarii (IC50 = 7.5 × 10−8M), and by clonidine in hippocampus (IC50 = 5 × 10−8M), spinal cord (IC50 = 4.5 × 10−8M), rostrolateral ventricular medulla (IC50 = 2.5 × 10−7M), and nucleus tractus solitarii (IC50 = 10−7M). This effect was counteracted by the selective α2-antagonists yohimbine and rauwolscine. A significant glutamate and aspartate release response was also induced by KCl (35 mmol/L) in hippocampus (+250 and +135%, respectively) and spinal cord (+120 and +55%, respectively), in vitro. However, neither mivazerol nor clonidine, at doses up to 10 µM, had any significant effect on KCl-induced glutamate release in spinal cord, whereas mivazerol blocked completely the release of both amino acids in hippocampus and only the release of aspartate in spinal cord. On the other hand, clonidine (1 µM) was only effective in reducing by 40% the release of aspartate in hippocampus. These data indicate that (1) inhibition of KCl-induced norepinephrine release by mivazerol is mediated by its action on α2-adrenergic receptors; (2) at concentrations selective for α2-adrenergic receptors, only mivazerol was effective in blocking the KCl-induced glutamate release in hippocampal tissue; and (3) at the same concentrations, both mivazerol and clonidine were unable to inhibit glutamate release in the spinal cord. These data suggest that prevention of hyperadrenergic activity by mivazerol in perioperative patients may be mediated through its effect on the release of norepinephrine and/or the release of glutamate and aspartate in regions of the CNS that are involved in the control of cardiovascular homeostasis.

Notes: Abstract: The cholinergic amacrine cells of the rabbit retina can be labeled with [3H]choline and the activity of the cholinergic population monitored by following the release of [3H]acetylcholine. It has been proposed that l-homocysteate may be the main endogenous transmitter released onto cholinergic amacrine cells by bipolar cells. Therefore, we have examined the effects of the isomers of homocysteate on the release of [3H]acetylcholine. In magnesium-free medium, d-homocysteate was slightly more potent than the l-isomer. The addition of magnesium, which blocks responses mediated by NMDA receptors, preferentially reduced but did not eliminate, the response to l-homocysteate. 2-Amino-7-phosphonoheptanoate, a potent NMDA antagonist, preferentially blocked l-homocysteate evoked responses. 6,7-Dinitroquinoxaline-2,3-dione, a potent kainate antagonist, preferentially blocked d-homocysteate-evoked responses. Therefore, in the rabbit retina, l-homocysteate is an NMDA-preferring agonist, whereas d-homocysteate is a kainate-preferring agonist. In addition, we found that l-homocysteate can activate the physiologically activated kainate receptor but only when used in millimolar concentrations and under conditions that minimize NMDA-receptor activation. However, the low potency of l-homocysteate combined with low affinity for the glutamate transporter, lack of immunocytochemical localization in bipolar cells, and low retinal content place serious limitations on the role of l-homocysteate at the bipolar-to-cholinergic amacrine cell synapse.

Notes: Abstract: Stimulation of synaptoneurosome suspensions by the neurotransmitter glutamate gives rise to rapid loading of ribosomes onto mRNA and increased incorporation of amino acids into trichloroacetic acid-precipitable polypeptides. Metabotropic glutamate receptors (mGluRs) are responsible for this effect. Although simultaneous Ca2+ entry and mGluR stimulation do not change the response, entry of Ca2+ 30 s or 3 min before mGluR stimulation markedly depresses the polyribosomal loading. Either NMDA or ionophore (A23187) produces the depression. A calmodulin antagonist, W7, alleviates the effect, suggesting that inactivation of phospholipase A2 by calcium-calmodulin-dependent kinase II is partially responsible for the phenomenon. Thus, interaction between different classes of glutamate receptors affects the control of protein translation at the synapse. This effect may partially explain recent observations of negative interactions between receptor classes in induction of long-term potentiation.

Notes: Abstract: Nitric oxide (NO), liberated from the photoactive donor Roussin's black salt (RBS), was investigated for its ability to release tritium from [3H]dopamine-loaded rat striatal slices. Our results show that illumination of RBS-pretreated striatal slices caused an increase in basal dopamine release, which was reduced by ∼73% in the presence of oxyhaemoglobin (10 µM), indicating that it was mediated by liberation of NO. The release was insensitive to removal of extracellular calcium yet was not due to gross cellular damage of the tissue, as there was no detectable increase in lactate dehydrogenase release. Chelation of intracellular calcium with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM; 10 µM) had no effect on the dopamine release stimulated by illumination of RBS-pretreated slices. The concentration of BAPTA-AM was adequate to chelate intracellular calcium because it inhibited release evoked by the calcium ionophore ionomycin (10 µM). Superfusion with zaprinast (10 µM) had no effect on RBS-induced dopamine release, suggesting that a mechanism independent of cyclic GMP is involved. This study indicates that NO has a stimulatory effect on striatal dopamine release in vitro that is independent of calcium.

Notes: Abstract: l-Glutamate, NMDA, dl-α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and kainate (KA) increased the release of somatostatin-like immunoreactivity (SRIF-LI) from primary cultures of rat hippocampal neurons. In Mg2+-containing medium, the maximal effects (reached at ∼100 µM) amounted to 737% (KA), 722% (glutamate), 488% (NMDA), and 374% (AMPA); the apparent affinities were 22 µM (AMPA), 39 µM (glutamate), 41 µM (KA), and 70 µM (NMDA). The metabotropic receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylate did not affect SRIF-LI release. The release evoked by glutamate (100 µM) was abolished by 10 µM dizocilpine (MK-801) plus 30 µM 1-aminophenyl-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466). Moreover, the maximal effect of glutamate was mimicked by a mixture of NMDA + AMPA. The release elicited by NMDA was sensitive to MK-801 but insensitive to GYKI 52466. The AMPA- and KA-evoked releases were blocked by 6,7-dinitroquinoxaline-2,3-dione (DNQX) or by GYKI 52466 but were insensitive to MK-801. The release of SRIF-LI elicited by all four agonists was Ca2+ dependent, whereas only the NMDA-evoked release was prevented by tetrodotoxin. Removal of Mg2+ caused increase of basal SRIF-LI release, an effect abolished by MK-801. Thus, glutamate can stimulate somatostatin release through ionotropic NMDA and AMPA/KA receptors. Receptors of the KA type (AMPA insensitive) or metabotropic receptors appear not to be involved.

Notes: Abstract: While studying the delivery of cytoplasmic proteins to the presynaptic terminals of CNS neurons, we discovered unique characteristics of one protein (p118) conveyed in slow component b (SCb) of axonal transport, the large group of proteins representing the cytoplasmic matrix. Alone among the SCb group, p118 coisolated with the synaptic junctional complex on biochemical fractionation of the radiolabeled synaptic regions. Purification and amino acid sequencing of this protein revealed it is most likely the guinea pig form of type I (brain) hexokinase (ATP:d-hexose 6-phosphotransferase, EC 2.7.1.1). Further biochemical treatments were consistent with this identity. The majority of type I brain hexokinase has been thought to be associated primarily with membranes, in particular the mitochondrial outer membrane. We found that the majority of type I hexokinase is transported toward the terminals at a rate at least 10 times slower than that exhibited by the maximal or average rate of mitochondria. This suggests that, in the axon, the enzyme exhibits transient or dynamic interactions with mitochondria that are moving more rapidly. It is not clear whether hexokinase binds exclusively to mitochondria, or also exhibits association with nonmitochondrial membranes. The unexpected enrichment of hexokinase during synaptic junctional complex purification may result from its strong association with the presynaptic membrane portion of the synapse.

Notes: Abstract: The function of the phosphoinositide second messenger system was assessed in occipital, temporal, and frontal cortex obtained postmortem from subjects with bipolar affective disorder and matched controls by measuring the hydrolysis of [3H]phosphatidylinositol ([3H]PI) incubated with membrane preparations and several different stimulatory agents. Phospholipase C activity, measured in the presence of 0.1 mM Ca2+ to stimulate the enzyme, was not different in bipolar and control samples. G proteins coupled to phospholipase C were concentration-dependently activated by guanosine 5′-O-(3-thiotriphosphate) (GTPγS) and by NaF. GTPγS-stimulated [3H]PI hydrolysis was markedly lower (50%) at all tested concentrations (0.3–10 µM GTPγS) in occipital cortical membranes from bipolar compared with control subjects. Responses to GTPγS in temporal and frontal cortical membranes were similar in bipolars and controls, as were responses to NaF in all three regions. Brain lithium concentrations correlated directly with GTPγS-stimulated [3H]PI hydrolysis in bipolar occipital, but not temporal or frontal, cortex. Carbachol, histamine, trans-1-aminocyclopentyl-1,3-dicarboxylic acid, serotonin, and ATP each activated [3H]PI hydrolysis above that obtained with GTPγS alone, and these responses were similar in bipolars and controls except for deficits in the responses to carbachol and serotonin in the occipital cortex, which were equivalent to the deficit detected with GTPγS alone. Thus, among the three cortical regions examined there was a selective impairment in G protein-stimulated [3H]PI hydrolysis in occipital cortical membranes from bipolar compared with control subjects. These results directly demonstrate decreased activity of the phosphoinositide signal transduction system in specific brain regions in bipolar affective disorder.

Notes: Abstract: As seizures in experimental models can be induced by the activation and suppressed by the inhibition of glutamate receptors, it is often proposed that a high extracellular glutamate level subsequent to excessive presynaptic release and/or altered glutamate uptake is epileptogenic. The purpose of this study was to ascertain the link between seizure activity and high extracellular glutamate. To assist the detection of any putative rise in extracellular glutamate during seizures, microdialysis was coupled to enzyme-amperometric detection of glutamate, which provides maximal sensitivity and time resolution. Electrical activity and field potential were also recorded through the dialysis membrane to confirm that epileptic activity was present at the sampling site. No increase in dialysate glutamate content was detected during picrotoxin-induced seizures, even when the K+ concentration in the perfusion medium was raised to 50% above that measured previously during paroxysmal activity. In addition, sustained inhibition of glutamate uptake by l-trans-pyrrolidine-2,4-dicarboxylate increased the extracellular glutamate level 〉20-fold but did not produce electrophysiological changes indicative of excessive excitation. These findings indicate that seizures are not necessarily accompanied by an increased extracellular glutamate level and that increased glutamatergic excitation in epilepsy may result from other abnormalities such as increased density of glutamate receptors, enhanced activation subsequent to reduced modulation, or sprouting of glutamatergic synapses.

Notes: Abstract: Activated microglia, often associated with neuritic amyloid plaques in the Alzheimer's disease brain, are likely to contribute to the progression of the disease process, e.g., by releasing neurotoxic reactive oxygen and/or nitrogen intermediates. In the present study, whether the amyloid β peptide (Aβ), the principal constituent of amyloid plaques, can stimulate microglial respiratory burst activity and/or microglial production of nitric oxide was examined. Using neonatal rat microglial cultures as a model, it was found that neither the spontaneous release of nitric oxide nor the lipopolysaccharide-induced production of nitric oxide was altered in cultures previously incubated with synthetic Aβ(1–40). for 24 h. In addition, no direct stimulatory effect of Aβ(1–40) on the respiratory burst activity was observed. Nevertheless, concomitant with an increase in the number of responsive cells, a profound priming of the phorbol 12-myristate 13-acetate-evoked production of superoxide anion was observed in Aβ(1–40)-treated cultures. Thus, both the maximal rate and the total phorbol 12-myristate 13-acetate-induced production of superoxide appeared to be statistically significantly higher as compared with untreated cultures. It is concluded that, as far as activation of the microglial respiratory burst is concerned, Aβ(1–40) may merely act as a priming rather than a triggering stimulus.

Notes: Abstract: The suitability of two-dimensional (2D) proton spectroscopy for monitoring, in vivo, the changes in levels of brain metabolites induced by cerebral ischemia was investigated in an experimental model of 30-min reversible ischemia induced by four-vessel occlusion in the rat. The resulting data were compared with those obtained by one-dimensional (1D) proton and phosphorus spectroscopy. Phosphorus spectra obtained during ischemia showed significant drops in levels of phosphocreatine (−73%), β-ATP (−60%), and intracellular pH (to 6.30) and an increase in inorganic phosphate level (905%). 1D and 2D proton spectra showed decreases in the N-acetylaspartate/creatine-phosphocreatine ratio that were not significantly different [−21% (1D) and −32% (2D)]. Similarly, the increases in lactate/creatine-phosphocreatine ratio were not significantly different [2,546% (1D) and 3,020% (2D)]. 2D spectroscopy also indicated a decrease in aspartate (−66%) and an increase in the inositol-choline derivative (+124%) pools during ischemia and an increase in alanine pool (+516%) during reperfusion. The glutamate-glutamine pool and taurine content did not change significantly during ischemia but decreased during reperfusion. The glucose level transiently decreased (−67%) during ischemia and increased immediately after (+261%). The levels of all the metabolites investigated returned to control values within 175 min after ischemia. 2D spectroscopy seems to be a reliable method of monitoring the changes in levels of cerebral compounds known to be involved in ischemia.

Notes: Abstract: Cultures of neonatal rat superior cervical ganglia (SCG) were used to test the hypothesis that the cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) control GTP cyclohydrolase I (GTPCH) gene expression and 5,6,7,8-tetrahydrobiopterin (BH4) content as traits of the noradrenergic phenotype. Treatment for 7 days with 1 ng/ml of LIF was found to produce the characteristic switch in the SCG neurotransmitter phenotype reported by others, as evidenced by a 60% decline in tyrosine hydroxylase (TH) activity and a 75% increase in choline acetyltransferase activity. This LIF treatment paradigm decreased BH4 levels in a concentration-dependent manner, with a maximal decline of 60% observed at 1 ng/ml. Analysis of the time course of this response indicated that LIF decreased BH4 levels by 60% following 3–7 days of treatment. Treatment of cultures with CNTF (2 ng/ml) resulted in a decline in BH4 levels that was of equal magnitude and followed the same time course as that produced by LIF. The LIF-dependent decline in BH4 levels resulted from a reduction in GTPCH enzyme activity, which decreased by 75% following 7 days of treatment. Nuclease protection assays of RNA extracted from cells treated for 7 days with 2 ng/ml of LIF or CNTF detected a 78–96% reduction in GTPCH mRNA content relative to β-actin mRNA content. Concomitant decreases in TH and GTPCH gene expression in response to LIF or CNTF demonstrate a coordinated regulation of gene expression for this BH4-dependent enzyme and the rate-limiting enzyme in the synthesis of its essential cofactor, BH4. Moreover, these results indicate that GTPCH gene expression in SCG neurons should be regarded as a trait of the noradrenergic phenotype.

Notes: Abstract: Preincubation of botulinum neurotoxin serotype A, B, or E with ganglioside GT1b was previously found to enhance adherence of botulinum neurotoxin to synapsin I and an ∼116-kDa bovine brain synaptosomal protein; in contrast, adherence to these two proteins by tetanus neurotoxin required preincubation with GT1b. We have now found that preincubation of the neurotoxins with ganglioside GD3 enhances their adherence to the ∼116-kDa protein more than that with GT1b. A purified preparation of the water-soluble ∼116-kDa protein was obtained from bovine brain synaptosomes by preparative column sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. N-Terminal amino acid sequences were obtained for two tryptic fragments of the ∼116-kDa protein. These sequences matched with the data bank sequences for β-adducin, a cytoskeletal protein. The carboxy-terminal tail region of adducin, but not the head region, was adhered to by the neurotoxins. Adherence of the neurotoxin to adducin and synapsin I may facilitate presentation of the neurotoxin to its specific substrate(s).

Notes: Abstract: The γ-aminobutyric acid type A receptor cDNAs encoding the α6 subunit homologues from chicken and goldfish have been cloned and sequenced. These proteins exhibit 83 and 75% identity, respectively, to the rat α6 polypeptide. In situ hybridization has demonstrated that, as in mammals, the avian and teleost fish α6 subunit genes are predominately expressed in cerebellar granule cells. Correspondingly, flunitrazepam-nondisplaceable binding of [3H]Ro 15-4513 (a benzodiazepine partial inverse agonist), which is a major characteristic of γ-aminobutyric acid type A receptors that contain the α6 polypeptide, is also mainly found for cerebellar granule cells of fish and chick. The conservation of this expression pattern suggests that γ-aminobutyric acid type A receptors possessing the α6 subunit are of fundamental importance for cerebellar function and that the corresponding gene regulatory elements, e.g., granule cell-specific enhancers, have also been conserved.

Notes: Abstract: The effect of docosahexaenoic acid (DHA) on neuronal survival was studied in cultured cells isolated from newborn rat retina. In vivo, the content of DHA in the retina increased nearly fourfold from days 2 to 12 after birth, whereas in retinal cells in culture it remained constant. Unlike amacrine cells, the photoreceptor cells in control cultures underwent a selective degeneration, starting at day 7, that led to their massive death by day 11. The addition of DHA at day 7 led to its active incorporation by the cultures, increasing from 6 to 21% of total fatty acids in cell lipids, and completely prevented photo-receptor cell death. When other fatty acids were tested, both neuronal fatty acid composition and photoreceptor death were the same as in control cultures. These results indicate that DHA is specifically required for the survival of retinal photoreceptors.

Notes: Abstract: The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q10 value of 2.0 between 37 and 25°C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis-Menten model, giving the following kinetic parameter values: Km = 127 nmol/mg of protein; Vmax = 0.39 nmol/min/mg of protein. p-Chloromercuribenzenesulfonic acid, a thiol-reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the Km value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.

Notes: Abstract: Measurements were made of the effects of muscarinic agents on endogenous levels of cyclic AMP and cyclic GMP, and the turnover of radiolabeled inositol phosphates in the abdominal nervous system of larval Manduca sexta. Cyclic AMP levels were increased by treatment with 3-isobutyl-1-methylxanthine or tetrodotoxin, but the muscarinic agonist oxotremorine-M and the muscarinic antagonist scopolamine had no consistent effects. In contrast, cyclic GMP levels were significantly increased by oxotremorine-M and by oxotremorine-M in the presence of 3-isobutyl-1-methylxanthine and tetrodotoxin but not in the presence of scopolamine. Using lithium to inhibit the recycling of inositol phospholipid metabolites in isolated nerve cords, we detected a small but consistent increase in inositol phosphate production by oxotremorine-M. The primary inositol metabolite generated during a 5-min exposure to oxotremorine-M co-eluted from ion-exchange columns with inositol-1-monophosphate, although other more polar metabolites were also detected. This agonist-evoked increase in inositol phosphate production was unaffected by tetrodotoxin but inhibited by scopolamine, suggesting that it is directly mediated by muscarinic receptors. Further evidence for coupling between muscarinic receptors and inositol metabolism was obtained using a cell-free preparation of nerve cord membranes labeled with [3H]inositol. Incubation with oxotremorine-M evoked a significant increase in labeled inositol bisphosphate, consistent with muscarinic receptors coupling to phosphatidylinositol metabolism. The accumulation of inositol bisphosphate in cell-free preparations suggests that the normal breakdown to inositol monophosphate requires cytosolic components. Together, these results indicate that muscarinic acetylcholine receptors in Manduca couple predominantly to the inositol phospholipid signaling system, although some receptors may modulate cyclic GMP.

Notes: Abstract: The nervous tissue-specific protein B-50 (GAP-43), which has been implicated in the regulation of neurotransmitter release, is a member of a family of atypical calmodulin-binding proteins. To investigate to what extent calmodulin and the interaction between B-50 and calmodulin are involved in the mechanism of Ca2+-induced noradrenaline release, we introduced polyclonal anti-calmodulin antibodies, calmodulin, and the calmodulin antagonists trifluoperazine, W-7, calmidazolium, and polymyxin B into streptolysin-O-permeated synaptosomes prepared from rat cerebral cortex. Anti-calmodulin antibodies, which inhibited Ca2+/calmodulin-dependent protein kinase II autophosphorylation and calcineurin phosphatase activity, decreased Ca2+-induced noradrenaline release from permeated synaptosomes. Exogenous calmodulin failed to modulate release, indicating that if calmodulin is required for vesicle fusion it is still present in sufficient amounts in permeated synaptosomes. Although trifluoperazine, W-7, and calmidazolium inhibited Ca2+-induced release, they also strongly increased basal release. Polymyxin B potently inhibited Ca2+-induced noradrenaline release without affecting basal release. It is interesting that polymyxin B was also the only antagonist affecting the interaction between B-50 and calmodulin, thus lending further support to the hypothesis that B-50 serves as a local Ca2+-sensitive calmodulin store underneath the plasma membrane in the mechanism of neurotransmitter release. We conclude that calmodulin plays an important role in vesicular noradrenaline release, probably by activating Ca2+/calmodulin-dependent enzymes involved in the regulation of one or more steps in the release mechanism.

Notes: Abstract: The existence of both nitric oxide synthase (NOS) immunoreactive interneurons and amino acid neurotransmitter-mediated nitric oxide (NO) release in the striatum suggests a role for NO in modulating striatal function. To explore the potential interaction between NO and dopaminergic neurotransmission, the NO-releasing agent (±)-S-nitroso-N-acetylpenicillamine (SNAP) was administered locally into the anterior medial striatum of chloral hydrate-anesthetized rats. SNAP, at 0.5, 1, and 2 mM concentrations, elevated striatal extracellular (EC) dopamine (DA) to 200 ± 42, 472 ± 120, and 2,084 ± 496%, respectively, above baseline levels. Perfusion with (±)-penicillamine (PEN, 1 mM), the non-NO-containing carrier component of SNAP, was ineffective, indicating that PEN is not responsible for SNAP-mediated DA release. Additional microdialysis experiments suggest SNAP-mediated DA release is not due to NO-induced neurotoxicity or blockade of the DA transporter. The DA-releasing effect of SNAP was attenuated under calcium-free conditions and abolished in rats pretreated with reserpine (5 mg/kg), implicating a calcium-sensitive vesicular-dependent release process. To determine the mechanism of SNAP-mediated DA release, the guanylyl cyclase (GC) inhibitor LY 83583 (100 µM) was administered 100 min before and during the SNAP pulse. LY 83583 elevated EC DA levels approximately fivefold and potentiated the DA-releasing effect of SNAP to 2,598 ± 551% above basal DA levels. Similar pretreatments with both the noncompetitive N-methyl-d-aspartate (NMDA) antagonist MK-801 (10 µM) and the competitive NMDA-receptor antagonist (±)-3-(carboxypiperazin-4-yl)propyl-1-phosphonic acid [(±)-CPP, 100 µM] blocked SNAP-mediated DA release. SNAP-mediated DA release was also significantly blunted by pretreatment and coperfusion with MgSO4 (10 mM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 µM) but not (+)-2-amino-3-phosphonopropionic acid (AP-3, 10 µM). These results suggest that NO releases DA via a calcium-sensitive vesicular-dependent process that is independent of GC activation. In addition, NMDA and kainate/(±)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated mechanisms are implicated in NO-induced DA release.

Notes: Abstract: The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron(III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.

Notes: Abstract: There is mounting evidence that at least some of the neurotoxicity associated with Alzheimer's disease (AD) is due to proteolytic fragments of the β-amyloid precursor protein (βAPP). Most research has focused on the amyloid β protein (Aβ), which has been shown to possess ion channel activity. However, the possible role of other cleaved products of the βAPP is less clear. We have investigated the ability of various products of βAPP to induce membrane ion currents by applying them to Xenopus oocytes, a model system used extensively for investigating electrophysiological aspects of cellular, including neuronal, signalling. We focussed on the 105-amino-acid C-terminal fragment (CT105) (containing the full sequence Aβ), which has previously been found to be toxic to cells, although little is known about its mode of action. We have found that CT105 is exceedingly potent, with a threshold concentration of 100–200 nM, in inducing nonselective ion currents when applied from either outside or inside the oocyte and is more effective than either βAPP or the Aβ fragments, β25–35 or β1–40. The ion channel activity of CT105 was concentration dependent and blocked by a monoclonal antibody to Aβ. These results suggest the possible involvement of CT105 in inducing the neural toxicity characteristic of AD.

Notes: Abstract: A new family of membrane phosphoproteins designated as P9, P12, P15, P16, and P20 with corresponding apparent molecular weights of 9K, 12K, 15K, 16K, and 20K was characterized from rat brain by using in vitro exogenous or endogenous phosphorylation and autoradiography. As the phosphorylation was selectively inhibited by the protein kinase C (PKC) inhibitor PKC19–31 or Ca2+-chelating reagents and again stimulated by the PKC activator phorbol 12,13-dibutyrate, these proteins are thought to be the natural PKC substrates. Because P12, P15, P16, and P20 were neutral proteins (pl 7.0) and specifically distributed in neuronal membranes, the new family of membrane-associated PKC substrate proteins was referred to as neutrinins. Neutrinins were widely distributed in rat brain, being especially plentiful in the spinal cord, medulla oblongata, cerebellum, and midbrain, relatively scanty in the cerebral cortex, but lacking in cytosol of brain areas and cell membrane preparations of peripheral tissues. The expression of the developmental changes of neutrinins has been monitored by the in vitro exogenous phosphorylation approach, i.e., adding purified PKC to a deactivated synaptosomal plasma membrane system. Levels of all the neutrinin proteins in rat cerebral cortex, as represented by P12, P15, and P16, showed an ontogenetic increase from the early postnatal days to the adult. This appears to be correlated with the commencement of synaptogenesis.

Notes: Abstract: Cotransmission of classic transmitters at the synapse has been mentioned for both the CNS and the PNS. Neuropeptide Y (NPY) is a cotransmitter in noradrenergic neurotransmission. In an attempt to understand the heteroregulation of norepinephrine (NE) and NPY biosynthesis, the present study was performed using radioimmunoassay of NPY and northern blotting of cDNA probes for characterization of NPY mRNA. Values of NPY-like immunoreactivity (NPY-ir) were elevated in the cerebrocortex from rats that received treatment with fusaric acid, an inhibitor of dopamine-β-hydroxylase, with a parallel decrease in NE. Similar results were also observed in rats treated with DSP-4, an alkylator of vesicles in noradrenergic nerve terminals. Moreover, cerebrocortical NPY-ir was reduced in rats receiving treatment with pargyline, an inhibitor of monoamine oxidase, with an elevation of catecholamine in parallel. Activity of NPY mRNA was modified by these drugs in a similar way. However, values of NPY-ir and NE in the cerebrocortex were not influenced by treatment with sodium nitroprusside or guanethidine at a dose producing hypotensive effect. Mediation of hypotensive reflex can thus be ruled out. The data obtained suggest that in vivo decrease of NE by drugs increases biosynthesis of NPY in the cerebrocortex of rats.

Notes: Abstract: Bidirectional communication occurs between neuroendocrine and immune systems through the action of various cytokines. Responses to various inflammatory mediators include increases in intracellular reactive oxygen species (ROS), notably, superoxide anion (O2−) and nitric oxide (NO•). Neurotoxicity mediated by NO• may result from the reaction of NO• with O2, leading to formation of peroxynitrite (ONOO−). ROS are highly toxic, potentially contributing to extensive neuronal damage. We, therefore, evaluated the effects of a variety of inflammatory mediators on the regulation of mRNA levels for manganese superoxide dismutase (MnSOD) and inducible nitric oxide synthase (iNOS) in primary cultures of rat neuronal and glial cells. To determine age-dependent variation of mRNA expression, we used glial cells derived from newborn, 3-, 21-, and 95-day-old rat brains. Interleukin-1β, interferon-γ (IFN-γ), bacterial lipopolysaccharide (LPS), and tumor necrosis factor-α showed significant induction of MnSOD in both glial and neuronal cells. However, only LPS and IFN-γ increased iNOS mRNA. These data demonstrate that these two genes are similarly regulated in two cells of the nervous system, further suggesting that the oxidative state of a cell may dictate a neurotoxic or neuroprotective outcome.

Notes: Abstract: The multisubunit γ-aminobutyric acid type A (GABAA) receptor is heterogeneous in molecular and pharmacological aspects. We used quantitative autoradiographic techniques to generate detailed pharmacological profiles for the binding of the GABAA-receptor ionophore ligand tert-[35S]butylbicyclophosphorothionate ([35S]TBPS) and its modulation by GABA and the GABAA antagonists bicuculline and 2′-(3′-carboxy-2′,3′-propyl)-3-amino-6-p-methoxyphenylpyrazinium bromide (SR 95531). Regional differences in the actions of bicuculline and SR 95531 were correlated with the expression of 13 GABAA subunits in brain as reported previously. In some brain regions SR 95531 reduced [35S]TBPS binding much more than bicuculline, as illustrated by high ratios of bicuculline- to SR 95531-modulated [35S]TBPS binding. This ratio correlated positively with α2-subunit mRNA levels. Binding that was equally affected by SR 95531 and bicuculline occurred prominently in regions with abundant α1 mRNA expression. The present findings thus reveal a novel pharmacological heterogeneity based on differences between α1 and α2 subunit-containing GABAA receptors. The data aid in developing GABAA-receptor subtype-specific antagonists and in establishing receptor domains critical for the actions of GABAA antagonists.

Notes: Abstract: Previous studies have revealed changes in immunohistochemical stains for calcium-binding proteins after manipulations that influence intracellular calcium. Cases have been revealed in which these changes in immunoreactivity were not correlated with changes in protein amounts. The present experiments examined whether these effects might be explained by changes in antiserum recognition due to calcium-induced changes in protein conformation. Calretinin, calbindin D28k, and parvalbumin incubated in high calcium were recognized by antisera better than when they were incubated in low calcium. Using a calbindin D28k antibody, it was shown that this effect occurs within physiological calcium concentrations. Formalin fixation of the proteins in the presence of calcium resulted in greater antibody recognition than did fixation of proteins in calcium-free states. The calretinin antiserum appeared to recognize a portion of the molecule previously shown to undergo calcium-dependent conformational changes. A calcium-insensitive antiserum was made to a different fragment of calretinin. These results indicate that some antibodies to calcium-binding proteins preferentially recognize particular calcium-induced protein conformations. Given the potential for wide fluctuations in neuronal calcium, the present results indicate that quantitative estimates of intracellular calcium-binding proteins obtained from immunohistochemical studies of neurons must be interpreted with caution.

Notes: Abstract: Myelinated CNS tissues from homozygous/hemizygous and heterozygous jimpy rumpshaker jprsh mutant mice were examined to determine the consequences on myelin structure of this mutation in the proteolipid protein (PLP) gene. Polyacrylamide gel electrophoresis and immunoblotting of brain homogenates confirmed that there was a decrease in PLP levels on the B6C3 genetic background onto which this gene was bred. We also observed an increase in level of a protein band that could correspond to the uncharacterized 10-kDa PLP previously reported in jprsh mice on an Rb(1.3) 1Bnr background. High-performance TLC and densitometry of lipids from brain homogenate and isolated myelin revealed a decrease in content of cerebrosides and sulfatides. Electron microscopy on optic nerves revealed that normal radial component is retained in jprsh myelin, further substantiating that PLP is not a component of this junctional complex. X-raydiffraction measurements on unfixed optic nerves showed that the jprsh period is 5–10 Å larger than normal. Moreover, jprsh optic nerve myelin was unstable, as evidenced by a continual increase in the period postdissection. jprsh myelin that was equilibrated at varying pH and ionic strength typically had a larger than normal period under all conditions (both swelling and compacting). Our findings thus demonstrate that the biochemical abnormalities in the jprsh mutant correlate with a wider periodicity and less stable packing of the myelin.

Notes: Abstract: Exposure of cerebellar granule cells to NMDA in culture at 5 days in vitro, when cells are not yet vulnerable to NMDA, evoked a pronounced reduction in NMDA receptor activity, measured by NMDA-induced 45Ca2+ influx, and counteracted the normal developmental increase in NMDA receptors. The effect was concentration and time dependent, the half-maximal effect being reached at about 45 µM and by 4–5 h. The decrease in NMDA receptor function was accompanied by a significant reduction in the protein level of the obligatory NMDA receptor subunit (NR) NR1. Both parameters remained at a low level as long as the agonist was present. However, receptor down-regulation was reversible, as receptor protein levels and NMDA responses were restored to control values upon NMDA removal, this process requiring protein synthesis. NMDA treatment also elicited a decrease in NR1, NR2A, and NR2B subunit messenger RNA (mRNA) levels. However, in comparison with NMDA receptor proteins, the decrease was faster, and NMDA receptor mRNA content recovered to control levels within 24 h in spite of the presence of NMDA. Concerning the mechanisms of agonist-induced regulation of NMDA receptor expression, it seems that protein kinase C-mediated protein phosphorylation is not involved, whereas inhibition of Ca2+/calmodulin-dependent kinase II/IV by KN-62 does depress NMDA receptor expression even in the absence of NMDA.

Notes: Abstract: The ability of mitochondrial Ca2+ transport to limit the elevation in free cytoplasmic Ca2+ concentration in neurones following an imposed Ca2+ load is reexamined. Cultured cerebellar granule cells were monitored by digital fura-2 imaging. Following KCI depolarization, addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) to depolarize mitochondria released a pool of Ca2+ into the cytoplasm in both somata and neurites. No CCCP-releasable pool was found in nondepolarized cells. Although the KCI-evoked somatic and neurite Ca2+ concentration elevations were enhanced when CCCP was present during KCI depolarization, this was associated with a collapsed ATP/ADP ratio. In the presence of the ATP synthase inhibitor oligomycin, glycolysis maintained high ATP/ADP ratios for at least 10 min. The further addition of the mitochondrial complex I inhibitor rotenone led to a collapse of the mitochondrial membrane potential, monitored by rhodamine-123, but had no effect on ATP/ADP ratios. In the presence of rotenone/oligomycin, no CCCP-releasable pool was found subsequent to KCI depolarization, consistent with the abolition of mitochondrial Ca2+ transport; however, paradoxically the KCI-evoked Ca2+ elevation is decreased. It is concluded that the CCCP-induced increase in cytoplasmic Ca2+ response to KCI is due to inhibition of nonmitochondrial ATP-dependent transport and that mitochondrial Ca2+ transport enhances entry of Ca2+, perhaps by removing the cation from cytoplasmic sites responsible for feedback inhibition of voltage-activated Ca2+ channel activity.

Notes: Abstract: The rat N-methyl-d-aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[3H]dichlorokynurenic acid labeled a single high-affinity site (KD = 29.6 ± 6 nM; Bmax = 19.4 ± 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to previous reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine ([3H]MK-801) was not detected in NR1-1a-transfected COS cells. Although immunofluorescent labeling of electroporated COS cells demonstrated that the NR1-1a protein appears to be associated with the cell membrane, neither NMDA nor glutamate effected an increase in intracellular calcium concentration in fura-2-loaded cells, suggesting that homomeric NR1-1a receptors do not act as functional ligand-gated ion channels. Therefore, COS cells appear to differ from Xenopus oocytes with respect to the transient expression of functional homomeric NR1 receptors. Although expression of NR1-1a is sufficient to reconstitute a glycine binding site with wild-type affinity for antagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca2+ and channel occupancy by MK-801, when expressed in this mammalian cell line.

Notes: Abstract: Changes in cerebral cortical adenine nucleotide and adenosine levels during 10-, 20-, or 40-min periods of four-vessel occlusion producing cerebral ischemia in rats and reperfusions of 10, 45, or 90 min were determined to evaluate the effects of ischemia duration on mitochondrial function. Substantial recovery was evident following 10 or 20 min of cerebral ischemia but not, however, after a 40-min period of ischemia. A secondary decline in the cortical levels of ATP became evident following 40 min of cerebral ischemia and 90 min of reperfusion. Longer periods of ischemia may be associated with a loss of adenosine, limiting the resynthesis of ATP during reperfusion. A separate group of rats, resuscitated with 100% O2, demonstrated a more rapid recovery of mitochondrial function compared with animals that received room air during reperfusion following 20 min of cerebral ischemia. No detrimental effects of 100% O2 were observed during the 90-min period of reperfusion, indicating that 100% O2 does not promote early mitochondrial dysfunction.

Notes: Abstract: This study examined the hypotheses that low-level lead (Pb) exposure would increase dopamine (DA) binding sites, would do so preferentially in nucleus accumbens, and that such effects would be modified by concurrent DA agonist treatment. D1-like and D2-like binding sites and the dopamine transporter (DT) were measured autoradiographically in caudate-putamen and nucleus accumbens of rats exposed from weaning to 0, 50, or 150 ppm Pb acetate drinking solutions with or without concurrent chronic intermittent intraperitoneal injections of the D1-like agonist SKF 82958 or the DA agonist apomorphine after 2 weeks (no injections), 8 months, or 12 months of Pb exposure. Pb selectively decreased DA binding in nucleus accumbens. Decreases in D2-like and DT sites were sustained across the 12-month exposure, whereas D1-like sites evidenced recovery at 12 months. Chronic intermittent DA agonist treatments reversed these effects of Pb in nucleus accumbens, restoring receptor and DT binding levels to normal, despite decreasing binding sites of non-Pb-treated rats. These studies implicate increased DA availability as a mechanism of Pb-induced DA system changes. They also raise the possibility that Pb exposure could serve as a predisposing factor in neurodegenerative diseases associated with DA system dysfunction or could alter the course of DA-based therapeutic treatments.

Notes: Abstract: Increasing evidence suggests that excessive activation of the calcium-activated neutral protease μ-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of μ-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of μ-calpain to its 80-kDa precursor was measured by western blotting. This μ-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24–48 h after injury. We also investigated the effect of μ-calpain activation on proteolysis of the neuronal cytoskeletal protein α-spectrin. Immunoreactivity for α-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of α-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of α-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of μ-calpain autolysis, calpain-mediated α-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although μ-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent μ-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.