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A model of ischemic-hypoxic brain injury which combines bilateral occlusion of common carotid arteries for 10 min and mild hypoxia (15% O2 for 10 min before and during occlusion) was developed. Global ischemia was assessed by a simplified EEG recording indicating isoelectric line, i.e. full arrest of cortical electrical activity. Histological examination of brain 7 days after ischemic insult showed from moderate to severe damage, mainly in the cerebral cortex (layers III, V and VI) and hippocampus (mainly CA1 subfield). The injury consisted of neuronal degeneration and necrosis with nuclear pyknosis and karyorrhexis. Immunohistochemical staining for gliofibrillar acidic protein showed a marked glial proliferation in the cerebral cortex and hippocampus. In the cortical slices, inositol phosphates accumulation stimulated by excitatory amino acid agonists (ACPD, ibotenate and quisqualate), as well as by norepinephrine and carbachol, was enhanced significantly (p0.01) with respect to sham-operated rats 7 days, but not 24 h, after the ischemic insult. The overall data show that the relatively simple transient brain hypoxia/ischemia rat model produces full arrest of cortical EEG, histopathological alterations and those relative to post-receptor neurochemical mechanisms characteristic of four-vessel occlusion model.

Ubiquitin (Ub) is a small 76-residue protein, involved in intracellular protein degradation through a specific ATP-dependent system, which uses Ub as a tag to label proteins committed to be hydrolyzed by a specific 26 S protease. PGP-9.5 is another important component of the Ub system, i.e. a neuron-specific carboxyl-terminal hydrolase, which recycles Ub from Ub-polypeptide complexes. We have investigated the expression of Ub and PGP-9.5 in rat hippocampal neurons in an early phase of reperfusion in a model of transient global brain ischemia/hypoxia (bilateral occlusion of common carotid arteries for 10 min accompanied by mild hypoxia—15% O2—for 20 min), by means of immunohistochemical methods using light and electron microscopy. The intensity of Ub and PGP-9.5 immunoreactivity was evaluated by image analysis. We have detected a marked increase of Ub immunoreactivity (UIR) in neurons of CA1, CA2, CA3, CA4, and dentate gyrus subfields 1 hr after ischemia/hypoxia (but not after hypoxia only), statistically significant as confirmed by image analysis. Such increase in immunoreactivity in ischemic/hypoxic rats was localized essentially in the nuclei of hippocampal neurons. There were no changes in PGP-9.5 immunoreactivity. The data suggest that in the present model of rat brain ischemia/hypoxia Ub is involved in the neuronal stress response.

The effects of metabotropic glutamate receptor (mGluR) agonists on inositol phosphates (IP) accumulation were investigated in slices of the cerebral cortex, hippocampus, striatum and cerebellum of adult Sprague-Dawley rats. EC(50) values for 1S, 3R-1-aminocyclopentane-1, 3-dicarboxylic acid (ACPD) did not differ significantly between various brain areas (range 10(-5) M), quisqualate was the most potent in all the brain areas (range 10(-7) - 10(-6) M), except the cerebellum (10(-5) M), ibotenate was the most potent in the striatum (range 10(-6) M) and the least potent in the cerebral cortex and hippocampus (range 10(-4) M). The efficacy in the four brain areas showed the following trend of ranking order for ACPD and quisqualate: hippocampusstriatumcerebral cortexcerebellum, and for ibotenate: hippocampuscerebral cortexstriatumcerebellum, although the observed differences reached the level of statistical significance only in the case of ACPD (hippocampus and striatum vs cerebellum) and ibotenate (hippocampus vs cerebellum). Co-incubation of the agonists at maximally effective concentrations in any pairwise combination resulted in no substantial additivity of IP accumulation. D,L-1-amino-3-phosphonopropionic acid (AP3) and D,L-2-amino-4-phosphonobutyric acid (AP4) at 0.5 mM concentration antagonized ACPD-induced IP accumulation by about 70 and 45 percent, respectively, without differences between brain areas. On the other hand, the antagonistic effects of L-serine-o-phosphate (SOP) at 1 mM concentration were the highest in the hippocampus (75 percent) and the lowest in the cerebellum (25 percent). The comparative data indicate considerable regional receptor heterogeneity, in terms of different ratios of response to the agonists (but not antagonists, except SOP). There is a robust responsiveness of mGluRs not only in the hippocampus and cerebral cortex, but also in the striatum which exhibits the highest affinity to both quisqualate and ibotenate.

The electric synaptic efficacy, in terms of extracellular electrical potentials, and the intracellular postsynaptic efficacy, in terms of inositol phosphate (IP) accumulation, were evaluated in rat hippocampal slices exposed for a brief period (10 min) to a high concentration of calcium (+2.7 mM). In addition, the effects of N -methyl- d -asparate (NMDA) ionotropic and metabotropic glutamate receptor (mGluR) antagonists on the induction and the establishment or maintenance of enhanced synaptic efficacy of CA1 pyramidal neurons due to high-calcium exposure were also tested. Elevation of the calcium concentration from 1.3–4 mM in the medium bathing hippocampal slices produced a long-lasting (60 over 90 min) increase in the slope of the CA1 somatic excitatory postsynaptic potential and the amplitude of the population spike (PS). Slice perfusion with NMDA antagonists cyclazocine and cis-4-phosphonomethyl-2-piperidine-carboxylic acid (CGS 19755) or with mGluR antagonists l -2-amino-3-phosphonopropionic acid (AP3) or alpha-methyl-4-carboxyphenyl-glycine (all 0.1 mM), during the 10-min period of exposure to high-calcium prevented the induction of such changes. By contrast, slice perfusion with the same concentration of CGS 19755 or l -AP3 did not affect the already established long-lasting increase in amplitude of CA1 PS induced by high-calcium. Moreover, high-calcium failed to produce any significant modification of the basal IP accumulation or of the IP accumulation elicited by mGluR agonist 1S,3R-trans-amino cyclo-pentane-1,3-dicarboxylic acid (ACPD). In conclusion, the results confirm that high-calcium induces a long-lasting increase in synaptic efficacy in rat hippocampal slices. Both NMDA ionotropic and mGluR receptors are involved in the induction, but not in the maintenance, of this phenomenon. In line with these data no modifications of basal or ACPD-induced phosphoinositide hydrolysis have been found during the maintenance stage.

The effects of 7-methoxytacrine (7-MEOTA), a less toxic derivative of tetrahydroaminoacridine, on the activity of acetylcholinesterase (AChE) molecular forms were investigated in vitro. AChE molecular forms were separated by sucrose gradient sedimentation from homogenates of the frontal cerebral cortex prepared with buffer containing Triton X-100 (soluble + membrane-bound enzyme). Two molecular forms, namely 10S and 4S corresponding to globular tetrameric (G4) and monomeric (G1) forms, respectively, were detected; their molecular weights were 220,000 and 54,000 Da. A significantly higher sensitivity to 7-MEOTA of G4 than of G1 forms was observed. The Ki values were 0.21 +/- 0.07 microM for the former and 0.70 +/- 0.15 microM for the latter. The differential inhibition of AChE molecular forms by 7-MEOTA is discussed in relation to its possible clinical application for treatment of disorders such as Alzheimer's disease, in which a reduction of brain cholinergic neurotransmission is believed to play a role.

The effects of a subacute intoxication with diisopropyl fluorophosphate (DPF) on total muscarinic acetylcholine receptor sites (mAChRs) and M-1 AChRs were evaluated in the cerebral cortex of young (2-4 months) and aged (22-24 months) Fischer 344 rats. Since M-1 AChRs are coupled to the metabolism of phosphoinositides, carbachol-induced accumulation of inositol phosphates (IP) and its inhibition by glutamate and NMDA was also measured in the cortical slices. DFP treatment caused about 75% inhibition of cholinesterase and 35% down-regulation of mAChRs (measured as [3H]quinuclidinyl benzylate binding) in both young and aged rats. The down-regulation of M-1-ACHRs (measured as [3H]pirenzepine binding) was more pronounced in aged (30%) than in young (17%) DFP-treated rats. There was a significant increase in carbachol-induced IP accumulation in aged, with respect to young, untreated rats. DFP treatment caused a considerable decrease in such IP accumulation in aged but not in young rats. Glutamate and NMDA antagonized carbachol-induced IP accumulation in untreated young and aged rats (and the effects of NMDA were reversed by carboxy-piperazinyl-propyl phosphonic acid). In DFP-treated rats such antagonism was somewhat less pronounced. The data appear of interest in relation to the use of anticholinesterase compounds in the therapy of senile dementia of Alzheimer's type. They suggest that beside their primary action (increasing brain ACh levels) such compounds also act on post-receptor mechanisms and on the interactions between cholinergic and glutamatergic neurotransmitter systems.

The effects of glutamate, NMDA and quisqualate on carbachol- and norepinephrine-elicited formation of inositol phosphate (IP) were evaluated in slices prepared from the cerebral cortex of 3- and 24-month Sprague-Dawley rats. Glutamate, NMDA, and quisqualate antagonized the IP response to carbachol in a concentration-dependent fashion. This antagonism was more pronounced in aged than in young rats, both for glutamate (IC5O 0.114 and 0.210 mM) and NMDA (IC5O 0.0029 and 0.127 mM), but not for quisqualate. Glutamate (but not NMDA) also antagonized in a concentration-dependent fashion the IP response to norepinephrine, IC50s were 0.061 and 0.126 mM for aged and young rats, respectively; quisqualate had an inhibitory effect only at 1 mM concentration in the two age-groups, while in aged rats some stimulatory effect was present at 0.1 mM concentration. Glutamate, NMDA and quisqualate (1 mM) did not affect basal IP accumulation in either young or aged rats; quisqualate, however, at 0.1 mM concentration had some stimulatory effect, more pronounced in aged rats. This effect was probably responsible for the biphasic effect of quisqualate in this age-group. The most important finding consists of the demonstration of an age-related increase in the inhibitory effects of NMDA on carbachol-induced IP accumulation. This implies an altered modulation of cholinergic post-receptor mechanisms by glutamatergic mechanisms.

A previous study conducted in this laboratory revealed a decrease in total cholinesterase (total ChE) in the cerebral cortex, hippocampus and striatum in aged rats (24 months) of various strains, as compared with young animals (3 months). The purpose of the present experiments was to extend the study to other brain areas (hypothalamus, medulla-pons and cerebellum) and to assess whether this decrease was dependent on the reduction of either specific acetylcholinesterase (AChE) or butyrylcholinesterase (BuChE) or both. By using ultracentrifugation on a sucrose gradient, the molecular forms of AChE were evaluated in all the brain areas of young and aged Sprague-Dawley rats. In young rats the regional distribution of total ChE and AChE varied considerably with respect to BuChE. The age-related loss of total ChE was seen in all areas. Although there was a reduction of AChE and, to somewhat lesser extent, of BuChE in the cerebral cortex, hippocampus, striatum, and hypothalamus (but not in the medulla-pons or the cerebellum), the ratio AChE/BuChE was not substantially modified by age. Two molecular forms of AChE, namely G4 (globular tetrameric) and G1 (monomeric), were detected in all the brain areas. Their distribution, expressed as G4/G1 ratio, varied in young rats from about 7.5 for the striatum to about 2.0 for the medulla-pons and cerebellum. The age-related changes consisted in a significant and selective loss of the enzymatic activity of G4 forms in the cerebral cortex, hippocampus, striatum, and hypothalamus, which resulted in a significant decrease of the G4/G1 ratio. No such changes were found in the medullapons or the cerebellum. Since G4 forms have been proposed to be present presynaptically, their age-related loss in those brain areas where acetylcholine plays an important role in neurotransmission may indicate an impairment of presynaptic mechanisms.

Previous studies in this laboratory showed an age-related decline of acetylcholinesterase (AChE) activity in the cerebral cortex of rats. In the present study the age-related differences in enzymatic activity were evaluated in terms of individual molecular forms. Extracts containing total, soluble and membrane-bound AChE were analyzed both by ultracentrifugation in sucrose gradient and by non-denaturing gradient polyacrylamide gel electrophoresis. By ultracentrifugation two molecular forms, namely 10S and 4S (corresponding to tetrameric-G4 and monomeric-G1 forms, respectively) were separated in extracts of total and soluble AChE, while only 10S forms were present in extracts of membrane-bound AChE. Electrophoresis of soluble AChE extracts revealed slowly- and fast-migrating bands, grouped in two clusters of at least three bands each; membrane-bound AChE contained only a single slowly-migrating band. Electrophoresis of the single forms isolated by ultracentrifugation showed that slowly- and fast-migrating bands corresponded to G4 and G1 forms, respectively. Therefore, in soluble AChE no one-to-one relationship between charge- and size-isomers was observed; on the contrary, such relationship has been shown for membrane-bound AChE. This implies that soluble G4 forms and membrane-bound-G4 forms are electrophoretically different, being heterogeneous the former and homogeneous the latter. The age-related decline of total AChE, accompanied by a decrease of G4/G1 ratio, depended mainly on a decrease of membrane-bound AChE while soluble AChE and its G4/G1 ratio was unchanged. The qualitative pattern of charge isomers was not modified by aging.

Potential age-related differences in the response of the ileum strip longitudinal and circular muscle to repeated treatment with diisopropyl fluorophosphate (DFP) were evaluated in Sprague-Dawley rats. The response was measured in terms of both biochemical parameters (acetylcholinesterase-AChE inhibition, muscarinic acetylcholine receptor binding sites-mAChRs, choline acetyltransferase-ChAT) and functional responsiveness (contractility of the isolated ileum stimulated by cholinergic agonists). The biochemical data were compared with those obtained for the cerebral cortex. Male 3- and 24-month old rats were s.c. injected with DFP on alternate days for 2 weeks (doses in mg/kg: first 1.1, two of 0.7 and four of 0.35). They were killed 48 hr and 7, 14, 21, 28 and 35 days after the last treatment. In the ileum strip of control rats there was a significant age-related decline of AChE, maximal density of 3H-QNB binding sites (Bmax) and ChAT. During the first week of DFP treatment the cholinergic syndrome was more pronounced in aged than in young rats, resulting in 35% and 10% mortality, respectively; subsequently the syndrome attenuated. At the end of DFP treatment ileal AChE were inhibited by about 30%; the down-regulation of mAChRs was about 50% in young and 35% in aged rats. No significant differences in the recovery rate of AChE were noted between young and aged rats (normalization within 7 days). On the contrary, mAChRs normalized within 5 weeks in young and 3 weeks in aged rats. This was probably due to more adaptive decline in the former group. There was a post-treatment increase of ChAT, transitory in young and persistent in aged rats. In spite of age-related marked loss of ileal mAChRs there were only little, although significant, changes in the contractile responsiveness of the isolated ileum to cholinergic agonists. Considerable DFP-induced down-regulation of mAChRs was not accompanied by changes in contractility stimulated by the agonists. The overall data indicate that age- and treatment-induced changes of AChE, mAChRs and ChAT in the ileum strip differ considerably from those observed in the cerebral cortex of the same rats.

Although ischemic stroke has higher incidence and severity in aged than in young humans, the age factor is generally neglected in ischemia animal models. This study was aimed at comparing age-dependent effects at early stages of transient global cerebral ischemia (TGCI) in rats. TGCI was induced in two groups of rats (3-6 and 20-24 months old, respectively) by exposure to 15% oxygen and 15 min occlusion of the two common carotid arteries. Brains were analysed in vivo by MRI-apparent diffusion coefficient (ADC) and T2 maps--at 1-3 h post-TGCI and in vitro by histochemical examination of triphenyltetrazolium chloride (TTC)-stained slices. At 1-3 h post-TGCI, a higher incidence of lesions was found in aged than in young rats especially in the hippocampus and cortex (occipital plus parietal) but not in the thalamus. The lesioned regions showed lower ADC values in aged than in younger rats. The most substantial ADC decreases were associated with enhanced spin-spin relaxation and lower TTC staining. The different responses of the two age groups support the use of aged animals for investigations on different ischemia models. Our model of brain ischemia appears appropriate for further studies including drug effects.

A model of transient global brain ischemia consisting of bilateral occlusion of common carotid arteries for 10 min and mild hypoxia (15% O2−85% N2) for 20 min was studied by means of MRI in young and aged Fischer 344 rats (3–4 and 24–26 months, respectively). Ischemia was assessed by full suppression of spontaneous EEG activity, which reappeared and normalized similarly in the two age-groups. The survival of young with respect to aged rats was considerably higher both at 24 h (20/20, i.e. 100% vs 12/16, i.e. 75%) and at 48 h (16/20, i.e. 80% vs 6/16, i.e. 38%). The localisation of brain lesions, their severity and progression were evaluated by a diffusion-weighted MRI (DWI) sequence at 24 and 48 h post-ischemia. There were no DWI-detectable lesions in eight out of 20 young and two out of 12 aged rats. The localisation of DWI-detected lesions was rather similar in rats of the two age-groups. In fact, the cerebral cortex, mainly parietal, occipital and temporal lobes were damaged in 83% of young and 90% of aged rats. The respective percentages for the thalamus were 83 and 60%, for the striatum 58 and 50%, and for the hippocampus 25 and 30%. The lesions present in the cerebral cortex and the thalamus were considerably more severe in aged than in young rats. In conclusion, in spite of similar localisation of ischemic lesions in the two age-groups, their incidence was higher, appearance more rapid and severity more pronounced in aged with respect to young rats. This resulted in a considerably higher mortality of the former. The overall data indicate that the age issue is very important in experimental ischemia research.

Previous study in this laboratory showed that following a sc injection of an organophosphorus compound, diisopropyl fluorophosphate (DFP), into rats the inhibition of 10S molecular forms was considerably more pronounced than that of 4S forms of brain acetylcholinesterase (AChE). This could depend on different accessibility of the two forms or on their different intrinsic sensitivity to the antiChE compound. In the present study the effects of DFP and Paraoxon on 10S and 4S forms were evaluated in vivo, i.e., after systemic administration, and in vitro by adding the organophosphorus compounds to each of the two forms after extraction from brain of untreated rats, solubilization and separation. The in vivo preferential inhibition of 10S forms was confirmed. The 10S/4S ratios for control and DFP-treated rats were 9.05 and 5.01, respectively; these ratios were 8.46 and 3.33 for Paraoxon. On the other hand, in the in vitro experiments there were no significant differences between IC50 values for 10S and 4S forms both in the case of DFP (2.66 and 2.98 microM) and Paraoxon (32.4 and 42.4 nM, respectively). The overall data suggest that the preferential in vivo inhibition of 10S molecular forms with respect to 4S forms depends on their different accessibility probably due to different subcellular localization of the two forms and not on their different intrinsic sensitivity.

Potential age-related differences in the recovery rate of brain cholinesterase activity (ChE) and muscarinic acetylcholine receptor binding sites (mAChRs) following reduction induced by repeated treatment with diisopropyl fluorophosphate (DFP) were evaluated in Sprague-Dawley rats. Male 3- and 24-month old rats were s.c. injected with DFP (doses in mg/kg: first 1.1, two of 0.7 and four of 0.35) on alternate days for 2 weeks and killed 48 hr and 7, 14, 21, 28 and 35 days after the last treatment. In the hippocampus and striatum, but not in the cerebral cortex, of control rats there was a significant age-related decline of ChE activity and maximal density of 3H-QNB binding sites (Bmax). The repeated administration of DFP during the first week caused a syndrome of cholinergic stimulation both in aged and young rats. The syndrome was more pronounced, in terms of intensity and duration (for many hours after each injection), in aged than in young animals resulting in 40 and 12% mortality, respectively; during the second week the syndrome attenuated in the two age-groups. The percentage inhibition of brain ChE at the end of DFP treatment (about 70%) did not differ between young and surviving aged rats. The down-regulation of mAChRs (without changes in affinity) was present in the three brain regions of both young and aged rats (from 20 to 40%). Factorial analysis of variance (2 ages x 2 recoveries ANOVA) showed significant differences for age, recovery rate, and significant interaction between age and recovery rate, both for ChE and mAChRs in the three brain areas. For example, cortical ChE in young rats reached pretreatment levels within 3 weeks, while hippocampal and striatal ChE activity recovered within 4 weeks; at these intervals ChE activity in aged rats was still considerably reduced (except in the striatum). Cortical and striatal mAChRs in young rats almost normalized within 1 week and hippocampal mAChR binding sites normalized within 2 weeks; at these intervals Bmax in aged rats were markedly below control levels. The overall data indicate that the recovery rate to normal baseline levels of ChE activity and mAChRs, following the termination of repeated treatment with the antiChE agent, is impaired in brain of aged rats. The delay in recovery rate is particularly evident in the cerebral cortex.

The effects of subchronic intoxication by diisopropylfluorophosphate (DFP) during pregnancy on development of brain cholinesterases (ChE) and [3H]quinuclidinyl benzilate (QNB) binding to muscarinic receptors were studied at the end of gestation and in offspring at 1, 5, 10, and 20 days after birth. Pregnant rats received on alternate days sc injections of DFP (first dose 1.1 mg/kg, subsequent doses 0.7 mg/kg) from Day 6 through Day 20. In spite of the considerable maternal toxicity of DFP, the level of brain ChE at birth and subsequent increases of enzymatic activity did not differ from those of controls. By contrast, significantly lower levels of QNB binding sites at birth were followed by a delay in development up to about 2 weeks. In the experiments using animals sacrificed on Days 20 and 21 of gestation, maternal brain ChE were consistently depressed, while in fetal brain an almost complete recovery occurred within 48 hr from the last DFP injection. Maximal number of QNB binding sites (Bmax) was decreased significantly both in maternal and fetal brain. These data indicate that tolerance mediated by receptor changes can be produced at early developmental stages, in spite of the accelerated recovery of brain ChE after treatment. In another experiment 7- to 27-day pups were treated on alternate days with sc doses of DFP (0.45 mg/kg from Day 7 to Day 19 and 0.70 mg/kg up to Day 27) and sacrificed on Day 7, 14, 20, 28, or 40. Brain ChE was reduced by 45–70%. Bmax of QNB binding sites showed a depression which became progressively more marked with increased duration of treatment, although the trend toward a progressive increase of receptor density as a function of age was preserved.

The effect of a single dose of diisopropyl fluorophosphate (DFP; Isoflurophate, 1.1 mg/kg s.c.) administered to rats during pregnancy was evaluated by measuring postpartum maternal and newborn brain-soluble and total acetylcholinesterases (AChE) and their molecular forms at intervals of 1, 2, 3, 4 and 10 days between treatment and sacrifice. Subsequently, the effects of DFP were studied in 18-day-pregnant rats, fetuses and placentae at 90 min and 24 h after treatment. The inhibition of postpartum maternal enzymatic activity did not differ from that previously found in adult males, while inhibition was considerably less pronounced in newborns at all time intervals, with a nearly complete recovery already at 48 h after treatment. An even faster recovery of brain enzyme was observed in 18-day fetuses from DFP-treated mothers (24-hour interval between treatment and sacrifice). In this experiment, a comparable inhibition was observed at 90 min after treatment in the adult and the developing brain, excluding a major influence of disposition factors in the differential recovery phenomena. An experiment on weanling rats yielded intermediate results between those of newborn and those of adult animals. Finally, most data confirmed previous findings that the soluble portion of brain AChE and medium molecular weight enzyme forms may have special significance in the initial phases of recovery.

The age-related changes in choline acetyltransferase (ChAT), cholinesterases (ChE) and muscarinic receptor sites (measured as Bmax of 3H-QNB binding) were evaluated in the cerebral cortex, hippocampus and striatum of Fischer 344 and Wistar male rats at the ages of 3 and 24 months. In the aged Fischer rats there was a significant decline of ChAT (except the hippocampus), ChE and muscarinic receptor densities in the regions analyzed. In the aged Wistar rats cortical and hippocampal ChAT as well as cortical muscarinic receptors remained constant while striatal ChAT, hippocampal and striatal muscarinic receptors decreased significantly; ChE were reduced in all regions analyzed. Factorial analysis of variance (2 strains x 2 ages ANOVA) showed significant strain-related differences in ChAT and muscarinic receptor sites in the three brain areas (about 1.5 times higher levels in the Fischer rats). The same analysis showed significant interactions between strain and age for ChAT and muscarinic receptors in the cerebral cortex, but not in the hippocampus and striatum; no interactions were found for ChE in the regions analyzed. This means that cortical ChAT and muscarinic receptors behave differently in aging in the two strains of rats, i.e., their alterations are strain-specific. Conversely, all other age-related changes (or lack of them for hippocampal ChAT) cannot be considered strain-specific. Moreover, an additional group of 33-month Wistar rats showed a significant decline of cortical muscarinic receptors with respect to 24 month rats but not of other markers in any area. The data underscore the need to consider genotype in the assessment of age-related cholinergic deficits in animal models.

Previous work in this laboratory showed that during intoxication of rats with diisopropyl fluorophosphate at day 20 of pregnancy the recovery of ChE activity was faster in fetal than in maternal brain. In the present study the differences between recovery rates in dam and fetus brain were evaluated in terms of molecular forms and spontaneous reactivation. Using ultracentrifugation on sucrose gradient two molecular forms of ChE, namely 10S (tetrameric globular G4 form) and 4S (monomeric G1 form) were detected both in maternal and fetal brain of untreated rats. The ratios 10S/4S were about 5.0 and 0.75 for dams and 20-day fetuses, respectively. DFP administration (1.1 mg/kg sc) inducing at 90 min an about 80% inhibition of ChE in maternal brain caused a shift in its 10S/4S ratio to 1.63, and to 0.53 in fetal brain (in which overall inhibition was about 70%). This means that 10S forms were preferentially inhibited by DFP both in maternal and fetal brain. After 24 and 48 hr there was a negligible recovery of overall ChE in maternal brain with no shift in the ratio. On the other hand, complete recovery of ChE in fetal brain within 48 hr was accompanied by almost total normalization of the 10S/4S ratio. Rapid recovery of fetal ChE appeared not to depend on hydrolysis of DFP-inhibited ChE. In fact, maternal and fetal DFP-inhibited enzyme preparations following the addition of oximes (pralidoxime or obidoxime) in vitro showed similar rates of reactivation. The overall data indicate considerable differences in recovery rate of molecular forms between dams and fetuses, but not in reactivation by dephosphorylation.

The spiny mouse ( Acomys cahirinus ) is the only precocial murid species. It has some neuroanatomical peculiarities such as a relatively thin cerebral cortex and a large hippocampus. The levels of choline acetyltransferase. membrane-bound acetylcholinesterase and muscarinic receptor sites (measured as [ 3 H]quinuclidynil benzilate binding) were assessed in the whole brain on days 1, 7, 14, 21, 28 and 80 (adult), and compared with those of Wistar rats of the corresponding ages. At birth choline acetyltransferase was significantly higher in spiny mice than in rats but the adult levels were similar, with an overall increase of about 5.2- and 14-fold for the former and the latter species, respectively. Membrane-bound acetylcholinesterase level and maximal density of muscarinic receptor sites in spiny mice were considerably higher at birth, in contrast adult levels were significantly lower than in rats with a respective overall increase of about 1.5- and over 4.5-fold. The high degree of maturity attained at birth by spiny mice partially depends on the long gestation period. However, if we consider postconception age, the maturation of choline acetyltransferase appears to be delayed at birth in the spiny mice, probably in relation to the lack of external stimulation during intrauterine life. In the cerebral cortex, hippocampus and striatum of adult spiny mice, when compared with the rats, there were similar levels of choline acetyltransferase but lower levels of membrane-bound acetylcholinesterase and, in the cerebral cortex, lower density of muscarinic receptor sites.

The unilateral injection of kainic acid into the nucleus basalis magnocellularis (NBM) resulted in an alteration of the distribution of acetylcholinesterase (AChE) molecular forms in frontoparietal cortex ipsilaterally to the lesion. The G 4 G 1 ratio fell from 5.4 ± 0.8 in contralateral to 3.0 ± 0.5 in ipsilateral cortex. The NBM lesion effect thus, mimicks, the loss of tetrameric G4 form reported for various brain cortical areas of Alzheimer's disease (AD) patients. The data support the suggestion that G4 form is enriched in presynaptic nerve terminals.

Pre-treatment of rats with chlorpromazine at a dose of 15 mg/kg i.p. 60 min before they were killed in potentiation of toxicity of physostigmine (0.5 mg/kg i.p., 20 min before killing) as evidenced by about a 2-fold increase in the inhibition of total ChE and BuChE in whole-brain preparations, as measured by a radio-isotope method. Chlorpromazine also enhanced the toxicity of dichlorvos (dimethyl-2,2-dichlorovinylphosphate) at a dose of 5 mg/kg i.p. 10 min before the rats were killed, but to a lesser extent than that of physostigmine, whereas it did not enhance it all when doses of dichlorvos (8 and 10 mg/kg) alone caused an inhibition of total ChE over 65%. Regarding the neurochemical mechanisms of potentiation of physostigmine effects by chlorpromazine, a study on discrete brain regions (cerebellum, medulla oblongata + pons, hypothalamus, striatum, midbrain, hippocampus, cerebral cortex) showed a considerable quantitative similarity of effects in various regions both as concerns total ChE and BuChE; potentiation of BuChE was slightly more pronounced than that of total ChE. Potentiation did not depend on possible antiChE effects of chloropromazine in vivo since the drug alone did not produce any inhibition of total ChE in whole brain and brain regions, and it produced only a slight inhibition of BuChE in the cerebellum, medulla oblongata + pons and midbrain, which could not explain the pronounced potentiation in all regions. The results suggest that the neurochemical mechanism of potentiation is not selectively linked to brain cholinergic systems but possibly linked to more general phenomena such as alterations in membrane permeability.

The inhibitory effect of six butyrophenone neuroleptics: haloperidol, triperidol, moperone, floropipamide, benperidol and droperidol on the activity of partially purified aspecific horse serum ChE and on purified Electrophorus electricus specific AChE was studied with AThCh as substrate using Ellman's spectrophotometric method. All butyrophenones were found to be inhibitors of ChE, the K i being in the range 10 −5 M and all followed a fully competitive type of inhibition. There were small differences in K i -values among various butyrophenones, while there were great differences in K i -values between the butyrophenones, the two phenothiazines and imipramine, the last three drugs being far more potent competitive ChE inhibitors ( K i 10 −6 –10 −7 M) than the butyrophenones. All the butyrophenones were also found to inhibit AChE, the overall inhibitor constants being in the range 10 −4 to 10 −5 M and all showed a mixed type of inhibition. The most potent of the butyrophenones are droperidol and benperidol, the benzimidazolone derivatives. There are no great differences in the K i -values for the butyrophenones, the two phenothiazines and imipramine for inhibition of AChE.

The effect of triperidol on the synthesis of free acetylcholine in fresh tissue, on the activity of the choline acetylase system in extracts of acetone powders, and on the activity of acetylcholinesterase in homogenates was studied in various regions of rat brain. Triperidol at a concentration 0·2 mM decreases the free acetylcholine synthesis in brain cortex slices by about 80 per cent. This decrease, although considerably lower, is statistically significant even at a concentration of the drug of 0·002 mM. Triperidol is without effect on the activity of the choline acetylase system in cerebral cortex at concentrations varying from 0·001 mM to 0·2 mM. Triperidol is without effect on the activity of acetylcholine esterase in all regions studied at concentrations ranging from 0·001 mM to 0·2 mM.

The responsiveness of cerebral cortical α 1 -adrenoceptors and cholinergic muscarinic M 1 receptors was assessed in young (3 months) and aged (24 months) male Sprague-Dawley rats. The measure of responsiveness was the accumulation of inositol phosphate (IP) formed in [ 3 H] myo -inositol-preloaded cerebral cortical slices in the presence of lithium, following stimulation with various concentrations of noradrenaline (1–300 μM) and carbachol (5–1000 μM). In old rats the maximum response to noradrenaline was higher by 80%, and that to carbachol by 33%, indicating an increased responsiveness of the investigated receptors in senescence.

Anticholinesterases occupy a unique position in behavioral pharmacology and toxicology. In fact, the study of their effects and mechanisms of action has implications at widely differing levels. At one end, there is the problem of prevention and therapy of pesticide intoxication in man and animals, not to speak of the problems of production, storage, and disposal of chemical warfare agents. At the opposite end, investigators not directly concerned with these practical aspects have been using anticholinesterases as tools in the investigation of neurochemical and neuropsychological models, in order to elucidate the role of peripheral and central cholinergic systems.

The effect of dietary rapeseed oil (RSO) on hepatic hexobarbital metabolism has been studied in short-term experiments in mice by measuring both the activity of mixed function oxidase system in microsomal preparations in vitro and the in vivo hexobarbital-induced sleeping time. Moreover, the fatty acid pattern of liver tissue, with particular regard to erucic and gadoleic acids, was analyzed gas-chromatographically. The hexobarbital metabolism in young RSO-fed mice (RSO containing 52 % of erucic acid and 7.4 % of gadoleic acid; 50 cal% for 3 days) is lower by about 44 % than in controls treated in the same way with peanut oil, independently of sex. RSO did not have a statistically significant effect on hexobarbital metabolism in adult animals of both sexes. As to the dose-response study, the prolongation of the hexobarbital-induced sleeping time in young male mice is statistically significant when fed with 30 cal% in RSO for 3 days, while it is not more statistically significant in mice fed 15 cal% in RSO. In RSO mice (50 cal%) erucic and gadoleic acid account both for about 10 % of the total fatty acids in the liver tissue, while they are quite absent in controls. It causes a relative deficiency of linoleic and arachidonic acids, known to be essential for synthesis and maintenance of the membranes supporting hepatic microsomal enzymes responsible for the metabolism of some drugs.

Previous reports from this laboratory (9) showed a differential recovery of various forms of acetylcholinesterase (AChE) of the rat brain after poisoning by DFP, the recovery being particularly rapid for medium molecular weight forms, and the phenomenon markedly pronounced in the striatum (8). It is well known that DFP and paraoxon cause substantially the same symptoms in the early phase of poisoning although differing in their chemical structure, which determines the type of phosphorylated AChE formed (diisopropyl- and diethylphos-phoryl-, respectively) and the rate of subsequent recovery in vivo and in vitro (for references see 1,4,7,12).

During chronic intoxication by diisopropyl fluorophosphate (Isoflurophate, DFP; s.c. treatment on alternate days — first dose of 1.1 mg/kg, subsequent doses of 0.7 mg/kg each until the 23rd day) a partial recovery of enzymatic activity was found at 24 h after each DFP administration. Relative to maximal AChE depression at 90 min, these rises were more pronounced in the soluble portion of the enzyme than in total enzyme preparation, i.e., that containing mainly membrane-bound AChE. Moreover, from the 2nd DFP administration on, there was a persistent increase of medium-molecular-weight forms both in soluble and in total AChE. The results suggest an important role of the soluble portion of AChE and of medium forms in the process of recovery of enzymatic activity.

Oral administration to mice of ethylenethiourea (ETU) at single doses from 50 to 600 mg/kg caused a dose-dependent increase (up to 200 mg/kg) in hepatic microsomal aniline hydroxylase (AH) without affecting aminopyrine-N-demethylase activity or total microsomal cytochrome P-450 content. Maximal (2.4 fold) enzyme increase was observed 24 h after treatment by an ETU dose of 200 mg/kg and was followed by a return to control levels within 4 days. Pretreatment of mice with actinomycin D completely prevented the increase of AH, while the addition in vitro to the incubation mixture of ETU at concentrations up to 1 mM did not cause any changes of enzymatic activity. These data speak in favour of an induction of AH via de novo protein biosynthesis. A more detailed analysis of the phenomenon indicated that the stimulatory effect of ETU on AH did not add to that produced by β-naphthoflavone, but added to that due to phenobarbital. Therefore ETU and β-naphthoflavone appear to affect similar mechanisms inducing aniline hydroxylase activity.

The pharmacological and toxicological properties of such agents as muscarine, pilocarpine, physostigmine, arecoline, atropine, scopolamine, and nicotine were known for a long time before the substances were identified and the properties of cholinergic mechanisms assessed by modern physiological and biochemical methods (Holmstedt, 1975). In particular, the bizarre mixture of “stimulant” and “depressant” effects caused by the consumption of antimuscarinic-containing plants, such as Atropa belladonna (the deadly nightshade) or Datura stramonium (Jameston or Jimson weed, stinkweed, thorn-apple, devil’s apple, yerba del diablo), have stimulated the imagination of people in widely different cultures. This is reflected in the lore of older pharmacological and toxicological treatises, as shown by the following description of the effects of the consumption of belladonna berries (the description applies to a group of soldiers who ate the poisonous material in order to refresh themselves at the end of a tiring day): Some of them died on the spot, others were severely poisoned; most of them became cheerful and playful; they pushed and pinched each other; some looked idiotic. Visual hallucinations were observed: one of the soldiers, mistaking his own finger for his pipe, made efforts to light it with a burning piece of wood, without showing any symptom of pain; when somebody pushed back his arm, with a stupid look and a naive smile he rubbed the brand on his trousers made of white cloth, which took fire. A sergeant mistook a carriage for a cellar or a bread store, and pretended to distribute victuals to the troops; but he was alone. Other soldiers knelt down and tried hard to collect blades of grass and twigs which they put together in bundles. They said this was their fatigue duty, and started crying when they were obliged to leave it, since they feared punishment by the corporal.*

The effect of diazepam (5 mg/kg i.p.) on the toxicity of DFP was studied in mice untreated or treated with obidoxime (25 mg/kg i.p.). Diazepam significantly increased the LD50 of DFP from 3.9 to 5.2 mg/kg in the absence of obidoxime, and from 30.0 to 47.8 mg/kg in its presence. The possibility that this effect was due to diazepam interference with central cholinergic systems was investigated in terms of acetylcholine (ACh) brain levels. In the absence of obidoxime brain ACh levels significantly higher than controls were produced by DFP (3 mg/kg s.c), by diazepam (5 mg/kg i.p.) and by DFP + diazepam. In the presence of obidoxime the rise in brain ACh by DFP was totally prevented, as previously demonstrated for rats. Treatment with DFP, obidoxime and diazepam resulted in ACh levels significantly higher than either controls or DFP + obidoxime treated mice but significantly lower than after DFP alone. The results suggest that the mechanism of attenuation of DFP toxicity by diazepam is unrelated to central cholinergic systems.

Potential age-related differences in the response of Fischer 344 rats to subchronic treatment with diisopropylfluorophosphate (DFP) were evaluated in terms of brain cholinesterase (ChE) inibition and muscarinic receptor sites. Male 3- and 24-month old rats were sc injected with sublethal doses of DFP (first dose 1.6, subsequent doses 1.1 mg/kg on alternate days) for 2 weeks and killed 48 hrs after the last treatment. In the cerebral cortex, hippocampus and striatum of control rats a3 significant age-related reduction of ChE and of maximum number of H-QNB binding sites (Bmax) was observed. The administration of DFP to senescent rats resulted in more pronounced and longer lasting syndrome of cholinergic stimulation, with marked body weight loss and 60% mortality. The percentage inhibition of brain ChE induced by DFP (over 80% in all regions) did not differ between young and senescent rats. As expected, in young rats DFP caused a significant decrease of Bmax (without apparent changes in affinity), which in the cerebral cortex reached about 40%. In the surviving senescent rats, the percentage decrease of Bmax due to DFP with respect to age-matched controls was very similar to that of young animals, especially in the cerebral cortex. Thus, there is great variability in the response of aged rats to DFP treatment, from total failure of adaptive mechanisms resulting in death to considerable muscarinic receptor plasticity. The data support the view that the ability of central neurotransmitter systems to compensate for pathological or xenobiotic induced insult is an essential part of the aging process.

Treatment of rats with chlorpromazine (CPZ, 15 mg/kg i.p. 60 min before sacrifice) did not modify cholinesterase (ChE) activity, but considerably enhanced the inhibition of total ChE induced by physostigmine (PhS, 0.5 mg/kg i.p. 40 min 4fter CPZ) in brain, skeletal muscle, myocardium, lung, liver, and kidney. Additional experiments also showed a prolongation of PhS inhibition by CPZ in brain. The enhanced inhibition of total ChE due to CPZ depended in most peripheral organs on the effect on pseudoChE (as measured by a spectrophotometric method), except in the case of skeletal muscle in which potentiation of PhS effect was observed on true acetylcholinesterase (AcChE). The results indicate that the potentiation by CPZ of PhS inhibition occurs in all organs tested and is relatively non specific. CPZ was found to potentiate slightly the effects of Mevinphos but did not interact with Carbaryl, Diazinon or Azinphos. Furthermore, haloperidol did not potentiate the effects of physostigmine.

Lester and Crane1 found that while tissues of vertebrates contain mostly the Q 10-homologue of ubiquinone (coenzyme Q), certain insects, notably the house-fly, Musca domestica, and the cabbage white butterfly, Pieris rapae, are characterized by the presence of the Q 9 homologue, that is, a ubiquinone with a side-chain of 9, instead of 10, isoprenoid units.

1. The effect of triperidol on the metabolism of glucose, pyruvate, glutamate, aspartate and glycine was studied with rat brain-cortex slices, U-(14)C-labelled substrates and a quantitative radiochromatographic technique. 2. Triperidol at a concentration of 0.2mm decreased the oxygen uptake and the (14)CO(2) production by about 30% when glucose, pyruvate and glutamate were used as substrates, whereas no effects were observed with aspartate and glycine. 3. The drug did not alter qualitatively the metabolic pattern of the substrates. 4. Quantitatively, triperidol decreased the incorporation of (14)C from [U-(14)C]glucose and [U(14)-C]-pyruvate into glutamate, glutamine and gamma-aminobutyrate but not into lactate, alanine and aspartate. The overall utilization rates of glucose and pyruvate were decreased. The relative specific radioactivities of glutamate and aspartate were also decreased. 5. Triperidol increased the rate of disappearance of U-(14)C-labelled glutamate, aspartate and glycine from the incubation medium, and altered the distribution of their metabolites between medium and tissue. 6. No appreciable effect of triperidol on [1-(14)C]galactose disappearance was found.

The reactivator obidoxime given twice i.p. at a dose of 12.5 mg/kg shortly before and after DFP (1.5 mg/kg s.c.), or given only once at a dose of 25 mg/kg shortly after DFP, prevented the rise of rat brain acetylcholine (ACh) observed 90 min after the anticholinesterase (antiChE) agent. At 180 min and 16 hr after DFP the brain ACh levels in the DFP and DFP-obidoxime groups were similar to that of the controls. The DFP-obidoxime groups did not differ from the corresponding DFP groups in regard to brain cholinesterase (ChE) activity (about 35 per cent of normal at 90 min, about 40 per cent at 180 min and about 65 per cent at 16 hr). The reactivation of ChE by obidoxime was complete in erythrocytes and partial in the serum. On the contrary, obidoxime did not modify DFP effects on liver, kidney and diaphragm ChE, in spite of a very high concentration of obidoxime found in the kidney.

Triperidol (TP) rapidly depresses the postganglionic response evoked by preganglionic stimulation of the superior cervical ganglion (s.c.g.) isolated in vitro , under sustained activity. Examination of total acetylcholine (ACh) by bioassay in the s.c.g. and in the incubation medium showed a TP-induced increase of ganglionic ACh coupled with a decrease in the medium. Parallel effects were observed on the labelled-ACh content of ganglia incubated with 3 H-choline. These effects are all consistent with the idea that a primary action of TP is to inhibit the release of ACh.