Janet R. Wetherell

Neurotransmitter Changes in Guinea‐Pig Brain Regions Following Soman Intoxication

Abstract: The effects of the organophosphate acetylcholinesterase (AChE) inhibitor soman (31.2 μg/kg s.c.) on guinea‐pig brain AChE, transmitter, and metabolite levels were investigated. Concentrations of acetylcholine (ACh) and choline (Ch), noradrenaline (NA), dopamine (DA), 5‐hydroxytryp‐tamine (5‐HT), and their metabolites, and six putative amino acid transmitters were determined concurrently in six brain regions. The brain AChE activity was maximally inhibited by 90%. The ACh content was elevated in most brain areas by 15 min, remaining at this level throughout the study. This increase reached statistical significance in the cortex, hippocampus, and striatum. The Ch level was significantly elevated in most areas by 60‐120 min. In all regions, levels of NA were reduced, and levels of DA were maintained, but those of its metabolites increased. 5‐HT levels were unchanged, but those of its metabolites showed a small increase. Changes in levels of amino acids were restricted to those areas where ACh levels were significantly raised: Aspartate levels fell, whereas γ‐Aminobutyric acid levels rose. These findings are consistent with an initial increase in ACh content, resulting in secondary changes in DA and 5‐HT turnover and release of NA and excitatory and inhibitory amino acid transmitters. This study can be used as a basis to investigate the effect of toxic agents and their treatments on the different transmitter systems.

Physostigmine and Hyoscine Improves Protection Against the Lethal and Incapacitating Effects of Nerve Agent Poisoning in the Guinea-Pig

This study is drawn from a work programme aimed at developing improved medical counter measures for nerve agent poisoning. Guinea-pigs were administered pyridostigmine (5.1 microg/h) or physostigmine (4.7 microg/h) and hyoscine (1.94 microg/h) for 6 days via a subcutaneously implanted mini osmotic pump. Pyridostigmine inhibited red cell acetylcholinesterase (AChE) by 44.2 +/- 2.7% and plasma cholinesterase (ChE) by 29.9 +/- 1.8%. Physostigmine and hyoscine inhibited red cell AChE by 18.7 +/- 3.7% and plasma ChE by 44.1 +/- 3.1%. On day 6, animals were challenged with a lethal dose of tabun (GA; 125 microg/kg), sarin (GB; 51.2 microg/kg), soman (GD; 31.2 microg/kg), GF (50 microg/kg) or VX (11.25 microg/kg) administered by the subcutaneous route. Animals were closely observed for signs of poisoning. The time to the onset of signs of poisoning was similar for all the agents except for VX, which showed a delay compared to the other agents. Following pretreatment with either pyridostigmine or physostigmine and hyoscine most animals survived for 2-3 h following nerve agent administration. In contrast, only physostigmine and hyoscine prevented or reduced the duration of the signs of incapacitation and the temperature drop produced by all the agents. Pyridostigmine-pretreated animals showed little or no recovery from incapacitation prior to death. Physostigmine and hyoscine pretreatment provided statistically (P < 0.05) better protection against GB, GD and VX lethality (24 h) than pyridostigmine pretreatment and better protection against GA and GF lethality.

Continuous Administration of Low Dose Rates of Physostigmine and Hyoscine to Guinea-pigs Prevents the Toxicity and Reduces the Incapacitation Produced by Soman Poisoning

Abstract— A regime was developed, using mini‐osmotic pumps, for the continuous subcutaneous administration of low doses of physostigmine (12.1, 9.7, 4.85 and 2.43 μgh−1), in combination with hyoscine (1.94 or 0.39 μg h−1), to guinea‐pigs for up to 13 days. Physostigmine, in combination with hyoscine, inhibited plasma Cholinesterase, and red blood cell and brain acetylcholinesterase, in a concentration‐dependent manner, did not affect the normal growth rate of guinea‐pigs, and produced no obvious signs of poisoning. A dose rate of 4.85 μg h−1 physostigmine and 1.94 μg h−1 hyoscine was required to inhibit red cell acetylcholinesterase by 30% and brain acetylcholinesterase by 5–15%, with an accompanying plasma hyoscine concentration of 700–850 pg mL−1. There was an apparent decline in red cell acetylcholinesterase activity during the 13 days. Hyoscine levels were higher in the cholinergic‐rich areas of the brain than in the plasma. Continuous pretreatment (1 or 6 days) with physostigmine (4.84 μg h−1) and hyoscine (1.94 μg h−1) provided complete protection against the lethal effects, and minimized the incapacitation and weight loss produced by soman at a dose equivalent to the LD99 value. Following soman challenge, guinea‐pigs exhibited early signs of soman poisoning, but generally these signs of poisoning were minimal by 1–2 h. Extending the pretreatment time to 13 days protected 75% of the guinea‐pigs against the lethal effects of soman poisoning. Red cell acetylcholinesterase activity, 24 h after soman poisoning, was higher following continuous pretreatment with physostigmine and hyoscine than after acute treatment with atropine.

A Comparison of the Distribution of Neurotransmitters in Brain Regions of the Rat and Guinea‐Pig Using a Chemiluminescent Method and HPLC with Electrochemical Detection

Abstract: Six brain areas of rats and guinea‐pigs, killed by microwave irradiation, were used for the concomitant measurement of the levels and regional distribution of cholinergic, biogenic amine, and amino acid neurotransmitters and metabolites. Acetylcholine (ACh) and choline (Ch) were quantified by chemiluminescence; noradrenaline (NA), dopamine (DA), 5‐hydroxytryptamine (5‐HT), and their metabolites by HPLC with electrochemical detection (HPLC‐EC); and six putative amino acid neurotransmitters by HPLC‐EC following derivatisation. The levels and regional distribution of these transmitters and their metabolites in the rat were similar to those reported in previous studies, except that biogenic amine transmitter levels were higher and metabolite concentrations were lower. The guinea‐pig showed a similar regional distribution, but the absolute levels of ACh were lower in striatum and higher in hippocampus, midbrain‐hypothalamus, and medulla‐pons. In all areas, the levels of Ch were higher and those of NA, 5‐HT, and taurine were lower than in the rat. The most marked differences between the rat and guinea‐pig were in the relative proportion of DA metabolites and 5‐HT turnover, as estimated by metabolite/transmitter ratios. This study can be used as a basis for a comprehensive understanding of the central effects of drugs on the major neurotransmitter systems.

Compared toxicity of the potassium channel blockers, apamin and dendrotoxin

The central toxicities of two potassium ion channel blockers, apamin and alpha-dendrotoxin (DTx), have been compared. Both apamin and dendrotoxin injected intracerebroventricularly produced signs of poisoning, including tremor and ataxia; however, only DTx produced changes in brain electrical activity, with high voltage spikes and epileptiform activity and subsequent brain damage. DTx, but not apamin, increased the amplitude of evoked field potentials and caused repetitive firing of neurones in hippocampal slices. Signs of poisoning following peripheral (intraperitoneal) administration of apamin were similar to those following central administration, including dramatic haemorrhagic effects on the lungs of decedent animals. These results are consistent with dendrotoxin being a centrally-active neurotoxin producing epileptiform activity and brain damage, whilst apamin produces its most significant pathology in the lung, possibly involving a neurogenic mechanism.

Effect of acute physostigmine-hyoscine pretreatment on the neurochemical changes produced by soman in the guinea-pig

The protective effects of two dose regimes of the organophosphate pretreatment combination, physostigmine and hyoscine, were assessed on the central neurochemical changes produced following soman intoxication. The lower dose combination (physostigmine 20 ?g/kg, hyoscine 10 ?g/kg, s.c.) inhibited brain regional acetylcholinesterase (AChE) by between 13.5 and 37.6% in all regions except the striatum, where there was no statistically significant inhibition. This low dose pretreatment failed to protect a measureable proportion of brain AChE from soman and did not prevent the neurotransmitter changes produced by soman. Signs of intoxication were indistinguishable from those seen with soman alone, however more animals survived longer than 24 h. The higher dose combination (physostigmine 196 ?g/kg and hyoscine 113 ?g/kg s.c.) inhibited brain regional AChE by between 56.8 and 67.3%, but had significant effects alone on the levels of ACh and NA. The high dose pretreatment effectively protected 20-30% of the enzyme following soman challenge. This protected enzyme became available as the physostigmine was eliminated from the body after 60-120 min. Animals exhibited mild signs of poisoning, such as hyperactivity and chewing, during the first 30-60 min, after which they recovered. Transmitter changes following soman were completely prevented with high dose pretreatment. Both dose levels of pretreatment decreased lethality but only the high dose, which protected a measurable amount of AChE and prevented neurotransmitter changes, decreased incapacitation.

Differential recovery of acetylcholinesterase in guinea pig muscle and brain regions after soman treatment

1 In brain areas of untreated guinea-pigs the highest activity of acetylcholinesterase was seen in the striatum and cerebellum, followed by the midbrain, medulla-pons and cortex, and the lowest in the hippocampus. The activity in diaphragm was sevenfold lower than in the hippocampus. 2 At 1 h after soman (27 mg/kg) administration the activity of the enzyme was dramatically reduced in all tissues studied. In muscle the three major molecular forms (A12, G4 and G1) showed a similar degree of inhibition and a similar rate of recovery and the activity had returned to normal by 7 days. 3 In the brain soman inhibited the G4 form more than the G1 form. The hippocampus, cortex and midbrain showed the greatest reductions in enzyme activity. At 7 days the activity in the cortex, medulla pons and striatum had recovered but in the hippocampus, midbrain and cerebellum it was still inhibited. 4 Thus the effects of soman administration varied in severity and time course in the different tissues studied. However the enzyme activity was still reduced in all tissues at 24 h when the overt signs of poisoning had disappeared.

A comparison of the decarbamoylation rates of physostigmine-inhibited plasma and red cell cholinesterases of man with other species.

Plasma and red cells from a variety of animal species were used to demonstrate that there is a relationship between the decarbamoylation rates of physostigmine-inhibited plasma and red cell cholinesterases in vitro and the effectiveness of carbamate pretreatment against nerve agent poisoning reported in the literature. Decarbamoylation rates were faster in the non-human primates than in the guinea-pig, and carbamate pretreatment is more effective in these species than in the guinea-pig. The data for the decarbamoylation rates of physostigmine-inhibited enzymes suggests that the non-human primates are the best animal model for extrapolation of protection studies from animal species to man. Control values for red cell acetylcholinesterase (AChE) activity (mumol/min/mL blood) using acetylthiocholine (1 mM) were higher in the human (4.98) and the rhesus monkey (4.14) than in the marmoset (0.84) and the guinea-pig (0.83). Plasma cholinesterase (ChE) activity (mumol/min/mL plasma) using butyrylthiocholine (10 mM) was highest in the rhesus monkey (9.29), intermediate in human (5.10) and guinea-pig (6.06), and lowest in the marmoset (4.07). There was a species difference in the relative activity of AChE: ChE in blood, human (65:35), rhesus monkey (45:55), marmoset (30:70) and guinea-pig (20:80). The rate of recovery of red cell AChE and plasma ChE activities, following incubation of whole blood with physostigmine (1 x 10(-7) M), was in the order human greater than rhesus monkey greater than marmoset greater than guinea-pig. During the incubation of red cells with physostigmine there was little recovery of AChE activity for 3-4 hr in any species. During the incubation of plasma with physostigmine there was complete recovery of ChE activity by 2-3 hr in the human and rhesus monkey and a significant recovery by 3 hr in the marmoset and guinea-pig. This suggests that a component of plasma, possibly ChE, was responsible for the degradation of physostigmine, presumably by hydrolysis. There was a marked species difference in the decarbamoylation rates of physostigmine-inhibited enzyme. In the red cell the t1/2 values (min) were 14.8 (human), 21.2 (rhesus monkey), 17.9 (marmoset) and 31.9 (guinea-pig). In the plasma the t1/2 values (min) were 11.2 (human), 32.9 (rhesus monkey), 44.1 (marmoset) and 52.4 (guinea-pig).

Effects of excitatory amino acid antagonists on dendrotoxin-induced increases in neurotransmitter release and epileptiform bursting in rat hippocampus in vitro.

Alpha‐dendrotoxin (α‐DTx), a snake venom toxin which blocks several types of fast‐activating voltage‐dependent potassium channels, induces limbic seizures and neuronal damage when injected into the brain. The mechanisms underlying these convulsant and neuropathological actions are not fully understood. We have studied the effects of α‐DTx on neurotransmitter release and electrical activity in rat hippocampal brain slices and the role of excitatory amino acid receptors in mediating these actions of the toxin. α‐DTx increased the basal release of acetylcholine, glutamate, aspartate, and GABA in a concentration‐dependent manner and induced epileptiform bursting in the CA1 and CA3 regions of the slice. The increase in neurotransmitter release was evident during the first 4 min after toxin addition, whereas the bursting appeared after a concentration‐dependent delay (20–40 min with 250 nM toxin). The N‐methyl‐D‐aspartate (NMDA) receptor antagonists AP5 and MK‐801 had no effect on the frequency or amplitude of dendrotoxin‐induced epileptiform bursts, but the non‐NMDA antagonists CNQX and DNQX abolished bursting in both CA1 and CA3 within 4–6 min. In contrast, the toxin‐induced increases in neurotransmitter release were not blocked by DNQX. This study has demonstrated that, following exposure to α‐DTx, there is a rapid increase in the release of neurotransmitters which precedes the onset of epileptiform bursting in the hippocampus. Since DNQX abolished the bursting but had no effect on the increase in neurotransmitter release, these results suggest that DNQX blocks α‐DTx‐induced epileptiform activity by antagonism of postsynaptic non‐NMDA receptors. J. Neurosci. Res. 48:499–506, 1997.

Structure-activity relations for anticholinergic 2(1-aryl(or cyclohexyl)-1-hydroxy-1-phenyl)methyl-1,3-dioxolans.

The syntheses and configurational assignments of some 4‐dimethyl aminomethyl‐2[1‐aryl (or cyclohexyl)‐1 ‐hydroxy‐1‐phenyl]methyl‐1,3‐dioxolans are described. The anticholinergic potency of the 4‐dimethylaminomethyl‐2[l‐cyclohexyl‐l‐hydroxy‐l‐phenyl]methyl‐1,3‐dioxolans, both in tertiary and quaternary form, depends principally on the configuration of the benzylic carbon atom, secondly on the C‐2 configuration and thirdly, and to a much lesser extent, if at all, on the C‐4 configuration. The dioxolans, which are derived formally from 4‐dimethylaminomethyl‐2‐methyl‐1,3‐dioxolan methiodide (or its tertiary analogue) by replacement of the 2‐methyl substituent by a 2[l‐aryl (or cyclohexyl)‐l‐hydroxy‐l‐phenyl]methyl group and the glycollates which are derived formally from acetylcholine (or its tertiary analogue) by corresponding substitution of the acetoxymethyl group have closely similar anticholinergic potencies.