Beth Hoskins

Effect of Acute and Chronic Cholinesterase Inhibition with Diisopropylfluorophosphate on Muscarinic, Dopamine, and GABA Receptors of the Rat Striatum

Abstract: The effects of acute and chronic administration of diisopropylfluorophosphate (DFP) to rats on acetylcholinesterase (AChE) activity (in striatum, medulla, diencephalon, cortex, and medulla) and muscarinic, dopamine (DA), and γ‐aminobutyric acid (GABA) receptor characteristics (in striatum) were investigated. After a single injection of (acute exposure to) DFP, striatal region was found to have the highest degree of AChE inhibition. After daily DFP injections (chronic treatment), all brain regions had the same degree of AChE inhibition, which remained at a steady level despite the regression of the DFP‐induced cholinergic overactivity. Acute administration of DFP increased the number of DA and GABA receptors without affecting the muscarinic receptor characteristics. Whereas chronic administration of DFP for either 4 or 14 days reduced the number of muscarinic sites without affecting their affinity, the DFP treatment caused increase in the number of DA and GABA receptors only after 14 days of treatment; however, the increase was considerably lower than that observed after the acute treatment. The in vitro addition of DFP to striatal membranes did not affect DA, GABA, or muscarinic receptors. The results indicate an involvement of GABAergic and dopaminergic systems in the actions of DFP. It is suggested that the GABAergic and dopaminergic involvement may be a part of a compensatory inhibitory process to counteract the excessive cholinergic activity produced by DFP.

Influence of chronic morphine treatment on protein kinase C activity: comparison with butorphanol and implication for opioid tolerance

The aim of this study was to determine whether chronic opioid treatment could influence the protein kinase C (PKC) activity in the rat brain. Chronic morphine (microns agonist) but not butorphanol (mu/delta/kappa mixed agonist) treatment enhanced cytosolic PKC activity in the pons/medulla, but not in the cytosolic fractions of cortex and midbrain regions. Concomitant administration of the opioid receptor antagonist, naloxone, blocked the PKC upregulation by chronic morphine. Chronic administration of morphine and butorphanol produced no change in the membrane PKC activity. Antinociceptive tolerance to morphine but not to butorphanol was developed under these conditions. These results suggest that chronic morphine administration leads to an upregulation of the cytosolic PKC activity in the pons/medulla through repeated activation of mu opioid receptors and that the PKC upregulation in this specific area may contribute to the morphine tolerance.

A protein kinase inhibitor, H-7, inhibits the development of tolerance to opioid antinociception

To investigate the effect of a protein kinase inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), on the development of tolerance to antinociception induced by morphine (mu-opioid receptor agonist) and butorphanol (mu/delta/kappa-mixed opioid receptor agonist), rats were infused i.c.v. with morphine, butorphanol and H-7 through osmotic minipumps for 3 days. Concomitant infusion of H-7 dose dependently inhibited the development of tolerance to i.c.v. morphine- and butorphanol-induced antinociception. These results suggest that protein kinases may play an important role in the development of opioid tolerance.

An assessment of comparative acute toxicity of diisopropyl-fluorophosphate, Tabun, Sarin, and Soman in relation to cholinergic and GABAergic enzyme activities in rats

The sc LD50s (mumol/kg) in rats for diisopropylfluorophosphate (DFP), Tabun, Sarin, and Soman were 14.5, 1.9, 1.4, and 0.88, respectively. The relative potency was as follows: DFP less than Tabun less than Sarin less than Soman (1:7.6:10.4:16.4). The relative potencies correlated with the in vitro acetylcholinesterase (AChE) inhibition (in terms of the IC50) by these compounds, in whole brain homogenates or the purified bovine erythrocyte AChE. There was a dose versus time for mortality relationship for all four compounds; the average time for death decreased with increase in dose. However, there was no correlation between time for death and the extent of AChE inhibition. The striatal as well as other regional (medulla, diencephalon, cortex, and cerebellum) AChE activity was inhibited over 90% of the control, by the lethal doses of these compounds. None of the lethal or sublethal doses had any apparent effect on choline acetyltransferase (CAT) or GABA-transaminase activities. Glutamic acid decarboxylase activity was increased by Soman, Sarin, and Tabun at certain lethal doses but was not affected by DFP even at the lethal dose. The results indicate that (a) the acute toxicity of organophosphate acetylcholinesterase inhibitors is directly related to the inhibition of AChE though there is a wide difference in their potency; (b) a substantial inhibition of AChE activity (over 90% of control) is necessary for lethality to ensue after an acute exposure and the margins between lethal and nonlethal doses are extremely small; and (c) qualitative differences seem to exist among the various organophosphates in affecting noncholinergic neurotransmitter enzymes.

Relationship between the neurotoxicities of soman, sarin and tabun, and acetylcholinesterase inhibition

Acetylcholinesterase (AChE)-induced chewing movements, tremors, convulsions and hind limb abduction at doses of 50-85% LD50 in rats were monitored in order to determine whether the severity of these different signs would correlate with brain AChE levels and the time course of such a relationship. 30 min after subcutaneous (s.c.) injection of Soman, the intensities of toxic signs were significantly correlated with the degree of striatal AChE inhibition. In the case of Sarin, the corresponding r-values were not significant except for tremors. For Tabun-induced chewing, tremor and hind-limb abduction, the r-values were significant. The neurotoxicity was most intense between 15 min to 2 h after treatment, but at 2 or 6 h, the r-values were well below 0.5. The inhibition of brain AChE was maximal by 30 min and was still high at 24 h.

A striatal serotonergic involvement in the behavioural effects of anticholinesterase organophosphates

Acute administration of the anticholinesterase agents diisopropylfluorophosphate, soman, sarin and tabun to rats in sub-convulsive doses produced tremors and hind-limb abduction. The ratios of the levels of 5-HIAA to 5-HT in the striatum showed a consistent tendency to be increased, suggesting an increase in 5-HT turnover. Both the behavioural and the biochemical changes were antagonized by atropine. The possible involvement of a cholinergic-serotonergic interaction in these tremors and hind-limb abduction is discussed.

Behavioral and neurochemical effects of continuous infusion of cocaine in rats

The ability of continuous intravenous infusion of cocaine (60 mg/kg per day for 11 or 12 days; by osmotic minipump) to alter responses to acute injection of cocaine (20 mg/kg, i.p.; given 24 hr after termination of the infusion by minipump) was tested in conscious, tethered Sprague-Dawley rats. Extracellular levels of cocaine, dopamine and metabolites of dopamine in the striatum were determined by in vivo microdialysis. Locomotor activity and stereotyped behavior were evaluated simultaneously during dialysis sampling. Prior infusion of cocaine blunted the ability of acute challenge with cocaine to increase the efflux of dopamine in the striatum, locomotor activity and stereotypy. Increases in extracellular levels of homovanillic acid in the striatum were significantly greater in cocaine-infused rats than vehicle-infused controls, both prior to and after acute injections of cocaine. However, no differences between these two groups were observed in levels of cocaine in the striatum after acute challenge. Extracellular levels of dopamine in the striatum correlated significantly (P less than 0.05) with stereotypy in both groups but with locomotor activity only in cocaine-infused rats. The results indicate that behavioral tolerance occurred after continuous intravenous infusions of cocaine, that this was correlated with neurochemical tolerance to acute cocaine challenge and that alterations in the metabolism of cocaine did not account for the observed behavioral responses.

Effect on striatal dopamine metabolism and differential motor behavioral tolerance following chronic cholinesterase inhibition with diisopropylfluorophosphate

Rats were treated with diisopropylfluorophosphate (DFP), using 1 or 2 mg/kg acutely, or with 1 mg/kg daily for 4, 14 or 28 days. Their behaviors and striatal dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) levels were studied. The behaviors: tremors, chewing-movements and hind-limb abduction induced by DFP increased in a steeply dose-dependent manner. Chronic treatment for up to 28 days produced biphasic patterns of change for all the behavioral parameters. Tremor occurred in a complex spectrum of slow to intense fast types. Except for chewing, tolerance developed for these parameters, but at different rates. After acute treatment striatal DA and DOPAC levels were altered and the DOPAC/DA ratios were consistently increased within about the first two hr, suggesting an increased turnover of DA. After chronic treatment for 4 and 14 but not 28 days, both DA and DOPAC levels were decreased without a change in their ratios. It is suggested that the changes in DA metabolism arose secondarily to an elevation of brain acetylcholine following cholinesterase inhibition. A prolonged change in the levels or turnover of DA could be responsible for increase of postsynaptic DA receptor density previously found by us [36], which might then partly mediate the behavioral tolerance to DFP.

Effects of nor-binaltorphimine on butorphanol dependence

Butorphanol has been shown to act on mu-, delta-, and kappa-opioid receptors. However, the relative involvement of different opioid receptor subtypes in butorphanol dependence is not known. In the present study, nor-binaltorphimine, a long-acting non-peptide kappa-opioid receptor antagonist, was employed to mask central kappa-opioid receptors before and during the induction of butorphanol dependence in rats, so that the involvement of kappa-opioid receptors could be elucidated. The results revealed that treatment with nor-binaltorphimine markedly blocked naloxone-precipitated withdrawal signs of escape behavior, teeth-chattering, wet shakes, ptosis, body weight loss, and hypothermia at all doses tested, and attenuated the withdrawal symptoms of forepaw tremors (24 nmol: P < 0.001) and diarrhea (12 nmol: P < 0.05; 24 nmol: P < 0.01). In contrast, nor-binaltorphimine had no effect on yawning, ejaculation, nor urination in butorphanol-infused rats undergoing withdrawal. Three days of butorphanol infusion significantly increased KD values (in the cortex and striatum), decreased Bmax (in the cortex only) of [3H]U-69,593 binding, and shifted Ki of nor-binaltorphimine against [3H]U-69,593 (4.5 nM) binding in the cortex by more than 10-fold. Treatment with nor-binaltorphimine blocked the effects of butorphanol on kappa-opioid receptors. It is therefore concluded that kappa-opioid receptors are involved in mediating escape behavior, teeth-chattering, wet shakes, forepaw tremors, ptosis, diarrhea, weight loss, and hypothermia in butorphanol-dependent rats undergoing withdrawal. Furthermore, kappa-opioid receptors become desensitized to agonists (in the cortex and striatum), down-regulated (in the cortex), and supersensitive to antagonists in butorphanol-dependent rats.

Opiate receptor characteristics in brains from young, mature and aged mice.

Age-related differences in opiate receptors were determined using young (1 month old), mature (3 and 8 months old) and aged (20 months old) mice. 3H-Dihydromorphine binding to mu-receptors in brain synaptic membranes consisted of two components: one with high affinity and one with low affinity. High affinity mu binding sites in membranes from young and aged mice had significantly less receptor densities and higher affinities than the mature mice. In the membranes from aged mouse brain, the affinity of low affinity binding sites for 3H-dihydromorphine was also significantly increased when compared to those in membranes from the 8-month-old group. Membranes from the young and aged groups revealed significantly higher affinity for binding of the kappa ligand, 3H-(-)ethylketocylazocine, than mature mice, which was not accompanied by any change in the density of the receptors. There was no change in either the number or affinity of the binding sites for 3H-(D-Ser2-Leu5)-enkephalinyl-Thr, the delta receptor ligand, among young mature and aged groups.