DFP-induced elevation of strength-duration threshold in hen peripheral nerve

Previous research has indicated that organophosphorus agents which induce a delayed neuropathy also elevate the strength-duration threshold during the progression of the neuropathy. To establish further the correlation between strength-duration changes and delayed neuropathy, the purpose of the present study was to investigate the biochemical and electrophysiological effects of two additional agents: diisopropylflourophosphate (DFP) and phosphonothioc acid, methyl-[2-(dimethylamino)-ethyl]O-ethyl ester (VX). DFP-treated hens exhibited clinical signs of toxicity at the time of electrophysiologic recording and biochemical analysis. DFP treatment also resulted in significantly elevated thresholds of the strength-duration curves of both the sciatic and tibial nerves. The VX-treated animals exhibited no clinical signs, despite aggressive dosing, and there were no significant changes in the electrophysiologic characteristics (conduction velocity, relative refractory period, strength-duration threshold) of either peripheral nerve. Acetylcholinesterase activities of the brain and skeletal muscle were significantly reduced in the VX- and DFP-treated hens, whereas creatine phosphokinase activities in these tissues were unaffected. These results are consistent with the view that elevation of the strength-duration threshold in peripheral nerves is among the earliest indicators of organophosphorus-induced delayed neuropathy.     

Study of low molecular weight heparin effect on the relation between anticoagulant activity and antithrombin III affinity.

Low molecular weight heparin (FR-860), and conventional unfractionated heparin (UF-heparin) were fractionated by rabbit antithrombin III (AT III)-Sepharose, and the effects of each affinity fraction on the coagulation and fibrinolytic activities were investigated. FR-860 was fractionated to no-affinity, low-affinity (LA) and high-affinity (HA) fractions, and UF-heparin to LA and HA fractions. The HA fractions showed higher activities regarding the prolongation of activated partial thromboplastin time, anti-factor Xa activity and antithrombin activity compared with those of LA. The HA and LA fractions exhibited the enhancement of heparin cofactor II (HC II) activity and fibrinolytic activity in a dose-dependent manner. These results suggest that the antithrombotic activity of FR-860 is exerted through AT III and other mechanism such as HC II-mediated system.     

Correlation of binding sites for diisopropyl phosphorofluoridate with cholinesterase and neuropathy target esterase in membrane and cytosol preparations from hen

To find new putative target(s) for organophosphorus induced delayed neurotoxicity (OPIDN), we investigated the biochemical and pharmacological characteristics of [3H] diisopropyl phosphorofluoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens. Specific [3H]DFP binding was determined by subtracting non-specific binding from total binding. The binding sites of [3H]DFP, an organophosphate that induces OPIDN, were found not only on membrane but also in cytosol. Reduction of subsequent ex vivo specific [3H]DFP binding by in vivo pretreatment with unlabeled DFP was found in cytosol, not membrane. The reduced binding lasted to the onset of OPIDN, especially in spinal cord. These results suggest that the specific DFP binding sites in cytosol, rather than on membrane, are the most important with regard to the initiation of OPIDN. Inhibitors of cholinesterase (ChE) and neuropathy target esterase (NTE) other than DFP did not affect specific [3H]DFP binding to either membranes or cytosol in vivo. Additionally, inhibition of the activities of these esterases by these compounds was not consistent with either the degree of inhibition of the [3H]DFP binding or a time-dependent manner of OPIDN. These results suggest that DFP binding site(s) involved in the initiation of OPIDN may be different from the active sites of ChE and NTE.     

Differentiation of neurochemical basis of stress-induced analgesia in mice by selective breeding

Random mated Swiss mice swam for 3 min at 20 °C. The animals with ≤10 s and 50 s postswim latencies on the hot plate (56 °C) were selected as progenitors of low (LA) and high (HA) analgesia lines, respectively. Gradual divergence of latency distributions and postswim hind paw flick latencies between the two lines was observed in successive generations. As shown by the tail flick test applied to the sixth offspring generation, postswim analgesia in the HA line was not only more pronounced but also lasted longer compared to the LA line and the unselected controls. Possible differentiation of the endorphin system activity in the course of selection is discussed.     

Altered expression of neurofilament subunits in diisopropyl phosphorofluoridate-treated hen spinal cord and their presence in axonal aggregations

Diisopropyl phosphorofluoridate (DFP) is an organophosphorus ester, which produces organophosphorus ester-induced delayed neuropathy (OPIDN) in hen and other sensitive species. A single dose of DFP (1.7 mg/kg, sc.) produces mild ataxia in 7-14 days in hens, which develops into severe ataxia or paralysis with the progression of disease. OPIDN is associated with axonal swellings and degeneration of axons. This study was carried out to investigate the expression of neurofilament (NF) subunits in the spinal cord of DFP-treated hens. Hens were treated with a single dose of DFP and sacrificed 1, 5, 10, and 20 days post-treatment. Western blot analysis showed increased expression of middle molecular weight neurofilament protein (NF-M), and decreased expression of high molecular weight (NF-H) and low molecular weight (NF-L) neurofilament proteins in the 2 M urea extracts of spinal cord particulate fraction. These changes were observed within 24 h of DFP administration and persisted for 10-20 days. Thus, there was increase in the stoichiometry of NF-M:NF-L in the spinal cord of DFP-treated hens. Immunoprecipitation, cross-linking, and two-dimensional polyacrylamide gel electrophoresis showed the presence of heterodimers, but not heterotetramers, in the hen spinal cord extract. Immunohistochemical staining revealed the presence of all three NF subunits in the cytoskeletal inclusions in DFP-treated hen spinal cord cross-sections. The results suggested that each NF subunit might be accumulated by a different mechanism in the axonal aggregations of DFP-treated hen.     

A comparative study of binding sites for diisopropyl phosphorofluoridate in membrane and cytosol preparations from spinal cord and brain of hens

Biochemical events in the initiation of organophosphorus induced delayed neurotoxicity (OPIDN) are not well understood. To find new putative target(s) for OPIDN, we investigated the biochemical and pharmacological characteristics of [3H] diisopropyl phosphorofluoridate (DFP) binding to membrane and cytosol preparations from the brain and spinal cord of hens in vitro. [3H]DFP binding to both preparations was determined by the specific binding obtained by subtracting non-specific binding from total binding. The specific binding sites of [3H]DFP were found not only on membrane but also in cytosol. Kd values were higher and Bmax values were lower in cytosol than in membrane. Moreover, the Kd values in both membrane and cytosol preparations from spinal cord were lower than those of brain. The Bmax values in membrane and cytosol were similar between brain and spinal cord. The specific binding to both preparations was markedly displaced by unlabeled DFP. The specific binding of DFP to the membrane was highly or partly displaced by organophosphorus compounds (OPs) or a carbamate, respectively. However, both the OPs and the carbamate had considerably weaker blocking effects on the specific binding of DFP to cytosol. None of the compounds known to interact with neuropathy target esterase (NTE) had a strong blocking effect on the specific binding of DFP to either membrane or cytosol. These results show that the specific binding of DFP to the membrane may be binding with cholinesterase (ChE). However, cytosol, especially in spinal cord, may have DFP binding sites other than ChE and NTE.     

Relationship of working memory and EEG to academic performance: a study among high school students

Some biological and behavioral elements which could explain differences between high and low academic attainment (HA/LA) students were identified. The qEEG of subjects under the 10-20 derivation system was recorded at rest and while completing a 3-back working memory (WM) task. While completing the task LA students showed more theta and total absolute potency at rest, and HA individuals showed more energy in delta and theta frequencies in frontal regions; LA students made a higher number of mistakes while executing the WM task with no differences in reaction time between groups. We conclude that a diminished WM capacity is present in LA students.     

Genetic influences on brain stimulation-produced analgesia in mice. I. Correlation with stress-induced analgesia

The analgesic effect of electrical stimulation of the periaqueductal gray matter (PAG) was studied in mice selectively bred for high and low stress-induced analgesia (HA and LA lines, respectively). The current intensity required for stimulation-produced analgesia (SPA) in LA mice was 5 times that for HA mice. Naloxone produced a 4-fold increase of SPA threshold in HA mice, but was ineffective in LA mice. These findings suggest that the differential responsiveness of these two lines to the analgesic effect of stress reflects a more general genetic modification of the efficacy and mechanism of their pain-inhibitory systems.     

Individual differences in behavior of inbred Lewis rats are associated with severity of joint destruction in adjuvant-induced arthritis

The aim of our study was to test the hypothesis that differences in behavioral characteristics are linked to severity of arthritis in association with neuro-endocrine and immune reactivity in an inbred strain of rats. Lewis rats were selected as high-active (HA) and low-active (LA) animals based on their exploratory activity in the open field. Subsequently, adjuvant-arthritis (AA) was induced in both groups. We observed no differences in the severity of inflammation as determined by paw swelling and redness. However, LA and HA animals differed in the severity of bone destruction as determined on radiographs taken on day 30 after induction of AA. LA rats had more osteoporosis, periostal new bone formation, and bone destruction than HA rats. There were no differences between HA and LA rats in corticosterone response after acute or chronic immune challenge. Splenocytes of LA rats had a lower mitogen-induced IL-10 and IFNgamma production during AA. Histological examination revealed more intense factor VIII staining in arthritic joints of LA animals, indicating more pronounced synovial angiogenesis. In addition, LA rats had higher plasma VEGF, an important angiogenic factor. Expression of RANKL, a crucial factor promoting bone resorption, was also higher in joints of LA animals. Our data demonstrate that activity in the open field, a behavioral trait, is associated with the severity of bone destruction in AA. Lower production of bone-protective cytokines and a higher rate of angiogenesis leading to more synovial proliferation may be responsible for the more severe joint destruction in LA animals.     

Tau proteins-enhanced Ca/calmodulin (CaM)-dependent phosphorylation by the brain supernatant of diisopropyl phosphorofluoridate (DFP)-treated hen: tau mutants indicate phosphorylation of more amino acids in tau by CaM kinase II

Diisopropyl phosphorofluoridate (DFP) produces organophosphorus ester-induced delayed neurotoxicity (OPIDN) in hen, human, and other sensitive species. A single dose of DFP (1.7 mg/kg, s.c.) produces mild ataxia in 7–14 days in hens, followed by progression to severe ataxia or paralysis. We studied the effect of DFP administration on Ca²⁺/calmodulin-dependent phosphorylation of tau proteins by the brain supernatants of control and DFP-treated hens. Brain supernatants from DFP-treated hens showed enhanced in vitro phosphorylation of htau40 and its various mutants, but no change in the two-dimensional phosphopeptide pattern, when compared to control hen brain supernatants. Analysis of tau mutants phosphorylated by brain supernatant and recombinant CaM kinase II α-subunit showed that (1) brain supernatant CaM kinase II is mainly responsible for the phosphorylation of Ser⁴¹⁶, (2) Ser³⁵⁶, but probably not Ser²⁶², is phosphorylated by CaM kinase II, (3) no amino acid between Lys³⁹⁵–Ala⁴³⁷ except Ser⁴¹⁶ is phosphorylated by CaM kinase II, (4) a number of amino acids in the tau molecule, which are phosphorylated by the brain supernatant in the absence of Ca²⁺/calmodulin are also mildly phosphorylated by CaM kinase II. The enhanced Ca²⁺/calmodulin-dependent phosphorylation of tau proteins by brain supernatant of DFP-treated hens that includes phosphorylation of a number of amino acids is likely to alter the functional properties of tau proteins in OPIDN. The hyperphosphorylated tau may destabilize microtubules, alter axonal transport, and result in degeneration of axons in OPIDN.     

Diisopropyl phosphorofluoridate (DFP) treatment alters calcium-activated proteinase activity and cytoskeletal proteins of the hen sciatic nerve

Diisopropyl phosphorofluoridate (DFP) produces delayed neurotoxicity (OPIDN) in hens that is characterized by peripheral and central axonal degeneration. DFP administration resulted in mCANP activity inhibition in sciatic nerve and significant decrease in total NF-H, phosphorylated NF-H, vimentin, GFAP, tubulin, and tau. The degradation of cytoskeletal proteins even in the presence of decreased CANP activity may be ascribed to the release of intracellular Ca²⁺, elevation of other proteinase activity, or modification of cytoskeletal proteins resulting in their increased susceptibility in OPIDN.     

Effect of acute organophosphorus exposure on 2', 3',-cyclic nucleotide 3'-phosphohydrolase activity in hen neural tissue

The effect of various organophosphorus (OP) compounds on 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNP) activity and the relationship to neurotoxic response was studied. Adult White Leghorn hens were dosed with either diisopropyl fluorophosphate (DFP), tri-o-cresyl phosphate (TOCP), leptophos, or paraoxon. CNP activity was determined utilizing crude homogenates or purified myelin preparations from whole brain, spinal cord, and sciatic nerves at maximal neurologic dysfunction, evaluated as locomotor impairment. Enzyme assays of CNP revealed a decrease in brain CNP activity at the time of maximal locomotor impairment after a single, oral dose of leptophos. Decreased CNP activity was also noted in spinal cord preparations from DFP-treated hens at the time of maximal locomotor impairment. Sciatic nerve CNP activity was not altered following treatment with OP-compounds. Paraoxon, a non-neurotoxic OP-compound, had little effect on neural tissue CNP activity. Neurotoxic OP-compounds, that cause secondary degeneration of myelin (Wallerian), may be associated with decreased brain and spinal cord CNP activity at the time of maximal locomotor impairment.     

Delayed neurotoxic effects of bis (1-methylethyl) phosphorofluoridate (DFP) in the European ferret: a possible mammalian model for organophosphorus-induced delayed neurotoxicity

This study examined the effects of the organophosphorus delayed neurotoxicant bis (1-methylethyl) phosphorofluoridate (DFP) on the central nervous system of the European ferret. Animals received subcutaneous injections of either 2 or 4 mg DFP/kg b.w. The extent of neuropathology was determined by the Fink-Heimer method, the activities of neuropathy target esterase (NTE) and cholinesterase (ChE) by enzyme assay methods, and the severity of clinical signs by a graded scale. In ferrets injected with 4 mg DFP/kg b.w., dense axonal and terminal degeneration were noted at 21 and 28 days post-DFP in the gracile, inferior vestibular, and lateral reticular nuclei, medial and dorsal accessory nuclei of the inferior olive, and in cerebellar folia I-IV. Degeneration was also noted in laminae VI-VII throughout most of the spinal cord and in the ventral motor nucleus at the level of the cervical enlargement. Both NTE and ChE activities were maximally inhibited at 6 hr post-dosing. NTE activity returned to control levels by 4 days while ChE activities reached control levels at 21 days. Clinical signs at 21 and 28 days post-DFP ranged from slight hindlimb weakness to severe ataxia or hindlimb paralysis. Less severe degeneration and clinical signs were noted in the animals exposed to 2 mg DFP/kg b.w. These findings indicate that the European ferret may be a model species for assessing the effects of organophosphorus delayed neurotoxicants.     

Brevetoxins bind to multiple classes of sites in rat brain synaptosomes.

The brevetoxins (PbTx series), neurotoxins produced by the marine dinoflagellate Ptychodiscus brevis, cause dose-dependent activation of the voltage-sensitive sodium channel (VSSC). Saturation binding studies employing adult rat brain synaptosomes suggest the existence of a high affinity/low capacity (HA/LC) and a second, lower affinity/higher capacity (LA/HC) class of binding site. LIGAND analysis of saxitoxin and brevetoxin saturation binding data yields a statistically identical Bmax for the brevetoxin high affinity/low capacity (HA/LC) site (1.9 +/- 0.98 pmol/mg protein) and for saxitoxin (1.72 +/- 0.78 pmol/mg protein; P less than 0.001). The stoichiometry of HA/LC brevetoxin binding and saxitoxin binding approaches 1:1. Covalent modification of synaptosomes with a brevetoxin photoaffinity probe preferentially blocks the HA/LC binding site. Hill plots of saturation binding data yield a coefficient of 1.0 +/- 0.02, demonstrating a lack of cooperativity between brevetoxin binding site classes. Kd and Bmax for toxin binding are independent of membrane polarity, intimating that the observed low affinity/high capacity (LA/HC) binding characteristics are not due to modification of the HA/LC site, and strongly argue for the presence of multiple brevetoxin binding site classes. Half-maximal binding at the LA/HC site, and strongly argue for the presence of multiple brevetoxin binding site classes. Half-maximal binding at the LA/HC site occurs at concentration ranges for which the brevetoxins allosterically modulate binding of other natural toxins to their specific sites.     

Inherited propensity for neuropathic pain is mediated by sensitivity to injury discharge.

We reported previously that injury discharge (ID), a burst of impulses fired following nerve injury, plays a role in triggering autotomy, a neuropathic pain-related behavior in rats. Here we affirm this link using two lines of rats, derived by selective breeding from the Sabra strain to express high (HA) or low (LA) levels of autotomy following hindpaw denervation. Blocking ID in HA rats before injury suppressed autotomy. Correspondingly, artificial prolongation of ID in LA rats just prior to neurectomy, increased autotomy. The autotomy in these HA and LA rats was like that of their Sabra ancestors. This suggests that the underlying basis for selection of these lines was differential sensitivity of the CNS to the effects of ID.     

Behavioral effects of d-cycloserine in rats: the role of anxiety level

It has been reported that the glutamatergic N-methyl-D-aspartate (NMDA) receptor is involved in stress responses and that anxiety is the primary response to stress. Although individual differences in anxiety levels of rats have been demonstrated by using the elevated plus-maze (PM) test, the role of NMDA receptor activity in such individuality of anxiety is not clear. Here, we examined whether low (LA) and high (HA) anxiety rats might respond differently to treatment with d-cycloserine (DCS), a partial agonist of the glycine binding site located on NMDA receptors. Male Wistar rats were screened by using the PM and divided into LA and HA subgroups. On the next day, these rats were again tested in the PM, 30 min after the treatment with DCS (5, 10, or 30 mg/kg ip). Five days later, the rats were subjected to a 2-day forced swim (FS) test, receiving the DCS treatment again 30 min before the second day session. The PM data showed that DCS had anxiogenic effects in LA but not HA rats. The immobility of LA or HA rats in the FS test was not affected by DCS. The results indicate that the behavioral effects of DCS depend on the anxiety level of rats and have task-dependent behavioral consequences, suggesting that glycine binding sites on NMDA receptors are involved in individual differences of anxiety level.     

Mechano- and thermo-sensitivity in rats genetically prone to developing neuropathic pain

By selective breeding, lines of rats were derived which consistently expressed high (HA) or low (LA) levels of autotomy following sciatic nerve injury, autotomy being a behavior pattern presumed to reflect the presence of neuropathic paraesthesias and pain. We report here that intact (unoperated) HA and LA rats differ in their responsiveness to cutaneous mechanical and thermal stimuli. Thus, the autotomy trait, which was identified by its expression under conditions of nerve injury, shares determinants with sensory processing channels in the intact animal.     

Behavioral and neurochemical consequences of multiple MDMA administrations in the rat: role of individual differences in anxiety-related behavior

Using the elevated plus-maze (EPM), Wistar rats can be distinguished into high (HA) or low anxiety (LA) subjects. These differences seem to reflect traits, since HA and LA rats vary also in other anxiety-dependent tasks, neurochemical mechanisms, and psychopharmacological reactivity, including lasting consequences after single treatment with 3,4-methylenedioxymethamphetamine (MDMA). Here, we tested whether multiple MDMA treatments also have subject-dependent effects. Based on routine EPM screening, male Wistar rats were divided into HA and LA sub-groups, which received five (i.e. multiple) daily injections of MDMA (5 mg/kg) or saline, followed by a test battery, including a challenge test with MDMA, a retest in the EPM, a novel-object test, and a final neurochemical analysis. Acutely, MDMA led to comparable hyperactivity in HA and LA rats. After multiple MDMA, behavioral sensitization was observed, especially in LA rats. Open arm time during the EPM retest (min 0-5) correlated with that of the initial one only in those rats, which had received a single injection of MDMA. Rats with multiple MDMA, especially LA-rats, showed more open-arm time and locomotion during the subsequent 5-10 min of the retest. In a novel-object test, rats with multiple MDMA, again especially LA subjects, showed more exploratory bouts towards the novel object. Neurochemically, multiple MDMA led to moderately lower serotonin in the ventral striatum, and higher dopamine levels in the frontal cortex as compared to single MDMA; these effects were also moderated by subject-dependent factors. Our data show that low-dosed multiple MDMA can lead to behavioral sensitization and outlasting consequences, which affect behavior in the EPM and a novel object task. Detecting such sequels partly requires consideration of individual differences.     

Acute and long-term consequences of single MDMA administration in relation to individual anxiety levels in the rat

Our previous work has shown that normal male Wistar rats can differ systematically in their behavioral response to the elevated plus-maze (EPM), where animals with high (HA) or low anxiety (LA) levels can be identified based on the percentage of time spent in the open arms. These animals also differ in other behavioral tests (e.g. active avoidance), and in their serotonin levels in the ventral striatum. Here, we tested whether such HA and LA rats might respond differently to the amphetamine analogue 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"). This drug can affect psychomotor activation and anxiety; effects which are probably due to its pronounced serotonergic and dopaminergic impacts in the rat brain. Based on a routine screening procedure in the plus-maze, male Wistar rats were divided into HA and LA sub-groups, in which rectal temperature was measured. Thirty minutes after the i.p. injection of MDMA (7.5 or 15 mg/kg) or vehicle, they were again tested in the plus-maze. During the next 3 weeks, the animals underwent further behavioral tests (plus-maze, open field, active avoidance, forced swimming) to test for possible long-term consequences of MDMA. Rectal temperature was found to be higher in LA than HA rats and was especially increased with the higher dose of MDMA (15 mg/kg). In the acute plus-maze test, the lower dose of MDMA led to an anxiogenic-like profile, whereas the higher dose led to an anxiolytic-like profile, both in HA and LA rats. Possible long-term consequences of MDMA were only tested with 7.5 mg/kg MDMA, since the 15 mg/kg dose led to a high level of lethality. The analysis of open field, plus-maze (performed after 9-12 days), and forced swimming behavior (performed after 20-21 days) did not provide indications for lasting effects of MDMA. In contrast, active avoidance learning was impaired in LA- but not HA-rats treated with MDMA. A single injection of MDMA does not only have acute effects on anxiety and psychomotor activation, but can also have some prolonged or delayed task-dependent behavioral consequences. The detection of such sequels can require that individual differences are taken into account and here, determining anxiety levels in the EPM seems to serve as a useful approach.     

The effect of word imagery on priming effect under a preconscious condition: An fMRI study

Semantic priming is affected by the degree of association and how readily a word is imagined. In the association effect, activity in the perisylvian structures including the bilateral inferior frontal gyrus, the left middle temporal gyrus, and the supramarginal gyrus was correlated. However, little is known about the brain regions related to the effect of imagery word under the preconscious condition. Forty word pairs for high (HA)‐, low (LA)‐, and nonassociation (NA), nonword (NW) conditions were presented. Each 40 association word pairs (HA and LA) included 20 high (HI) and 20 low (LI) imagery prime stimuli, using a visually presented lexical decision task. A trial consisted of 30 ms prime, 30 ms mask, 500 ms probe, and 2–8 s stimulus onset asynchrony. Brain activation was measured using functional magnetic resonance imaging during word discrimination. Behavioral data indicated that the shortest response time (RT) was given for HA words, followed by LA and NA, and NW showed the longest RT (P < 0.01). RT was faster in HI than LI within HA, but not LA conditions (P < 0.01). Functional neuroimaging showed that differential brain regions for high imagery (HI) and low imagery (LI) words within low prime‐target word association were observed in the left precuneus, left posterior cingulate gyrus, and right cuneal cortex. The present findings demonstrate that the effect of the degree of imagery on semantic priming occurs during the early stage of language processing, indicating an “automatic imagery priming effect.” Our paradigm may be useful to explore semantic deficit related to imagery in various psychiatric disorders. Hum Brain Mapp 35:4795–4804, 2014. © 2014 The Authors. Human Brain Mapping Published by Wiley Periodicals, Inc.