Age-dependent MRI-detected lesions at early stages of transient global ischemia in rat brain.

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.

Transient global brain ischemia in young and aged rats: differences in severity and progression, but not localisation, of lesions evaluated by magnetic resonance imaging.

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.

Transient global brain hypoxia-ischemia in adult rats: neuronal damage, glial proliferation, and alterations in inositol phospholipid hydrolysis.

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 (p < 0.01) 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.

Transient global brain ischemia in the rat: spatial distribution, extension, and evolution of lesions evaluated by magnetic resonance imaging.

A newly developed model of transient global ischemia in the rat was evaluated by magnetic resonance imaging (MRI) in terms of localization of brain lesions, their extent and severity, and temporal evolution. Such a model, consisting of bilateral occlusion of common carotid arteries for 10 minutes and mild hypoxia (15% O2) for 20 minutes induces delayed neuronal degeneration, necrosis, and gliosis (detected histologically and immunohistochemically). Ischemia was assessed by full suppression of spontaneous electroencephalographic activity. A "hybrid" T2-/diffusion-weighted MR sequence enhancing more effectively the contrast between injured and intact tissues as compared to T2-weighted MRI was used at 24, 48, 72, and 96 hours and at 7 days postischemia. Twenty hypoxic-ischemic rats showed a considerable variability in brain damage. In 8, there were no MRI-detectable lesions at any interval. In the other 12 rats, the severity and extension of neuronal damage varied markedly, but the lesions were always localized (monolaterally in 8 and bilaterally in 4 rats) in the occipital, temporal, or parietal cerebral cortex. Mainly, they were of intermediate severity or were severe (as assessed by MRI hyperintensity) and were accompanied by usually less severe lesions in the thalamus and/or caudate putamen. The hippocampus was affected moderately or severely in 4 of 12 rats. In most cases, there was at 48 hours a considerable growth in severity and/or extension of lesions, which usually remained stable at later intervals. In conclusion, MRI allowed us to follow brain lesions during the first week in this relatively simple and noninvasive model of transient global ischemia.

Ubiquitin-mediated stress response in a rat model of brain transient ischemia/hypoxia.

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.

Differential responsiveness of metabotropic glutamate receptors coupled to phosphoinositide hydrolysis to agonists in various brain areas of the adult rat.

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: hippocampus > striatum > cerebral cortex > cerebellum, and for ibotenate: hippocampus > cerebral cortex > striatum > cerebellum, 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.

Glutamate-dependent mechanisms in the induction of a calcium long-term potentiation-like phenomenon.

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 (80 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.

Differential inhibition of rat brain acetylcholinesterase molecular forms by 7-methoxytacrine in vitro.

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.

Differential reactivation by HI-6 in vivo of paraoxon-inhibited rat brain acetylcholinesterase molecular forms.

The effects of the cholinesterase reactivator HI-6, [1-(((4-(aminocarbonyl)-piridinio)methoxy)methyl-2-(hydroxy- imino)methyl pyridinium dichloride], on paraoxon-inhibited brain acetylcholinesterase (AChE) and its molecular forms were studied in rats. Treatment with paraoxon (0.25 mg/kg s.c.) caused approx. 60% inhibition of total AChE from frontal cerebral cortex, while that including HI-6 (140 mg/kg i.m.) and atropine (50 mg/kg i.m.) reduced such inhibition to only 25%. Two molecular forms of the enzyme, 10S and 4S, corresponding to globular tetrameric (G4) and monomeric (G1), were detected by sucrose gradient sedimentation. In paraoxon treated rats the G4 form was inhibited by approx. 65% while G1 only by 35%. The G4 form was considerably and selectively reactivated by HI-6 while the G1 form was not reactivated at all. The data show that HI-6 penetrates the blood-brain barrier and reactivates the molecular forms preferentially inhibited by paraoxon and involved in synaptic neurotransmission.

Carbachol-induced accumulation of inositol phosphates and its modulation by excitatory amino acids in cortical slices of young and aged rats with down-regulation of muscarinic M-1 receptors.

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.

Altered modulation by excitatory amino acids of cortical phosphatidylinositol system stimulated by carbachol in rats poisoned by an anti-cholinesterase compound, diisopropyl fluorophosphate.

The effects of glutamate and N-methyl aspartate (NMDA) on carbachol-induced inositol phosphate (IP) accumulation were evaluated in slices of the cerebral cortex of rats treated with diisopropyl fluorophosphate (DFP) for 2 weeks. This induced an about 75% inhibition of cholinesterases. The IP accumulation induced by carbachol (expressed as ratio stimulated/basal IP content) was lower in DFP rats than in controls when incorporation of [3H]-myoinositol into membrane phospholipids and their hydrolysis were measured (no washing step between labeling and hydrolytic incubation). There were no differences in carbachol induced IP accumulation between control and DFP rats when only phosphoinositide hydrolysis was determined (hydrolytic incubation of prelabeled washed slices). When both incorporation of [3H]-myoinositol and the hydrolysis were measured, 0.5 mM glutamate and 0.1 mM NMDA caused a significant, about 40%, decrease of carbachol-induced IP accumulation in control rats; the inhibitory effects of glutamate and NMDA were not significant in DFP rats. When only hydrolytic IP accumulation by carbachol was studied, the inhibitory effects of glutamate and NMDA were very similar in control and DFP rats. Additional experiments on inositol phospholipid synthesis showed a significantly lesser [3H]-myoinositol incorporation (by about 30%) in DFP rats. This may explain the differences between the results obtained by the two methods. The overall data suggest that the attenuation of glutamate and NMDA effects in DFP-rats depends on a decrease of carbachol-induced IP accumulation or phosphoinositide synthesis rather than on the EAA specific action.

Effects of excitatory amino acids on inositol phosphate accumulation in slices of the cerebral cortex of young and aged rats.

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.

Age-related changes in muscarinic receptor and post-receptor mechanisms in brain and ileum strip of rats.

Age-related differences in the response of the cerebral cortex and ileum strip to a repeated treatment with an anticholinesterase compound, diisopropyl fluorophosphate (DFP) were evaluated in 3- and 24-month Sprague-Dawley rats. The response was measured in terms of acetylcholinesterase (AChE) inhibition and total muscarinic receptor density (MAChRs, measured as 3H-QNB binding). At the end of DFP treatment there was a 75% inhibition of brain AChE and 30% inhibition of ileal AChE, independently of age. The adaptive down-regulation of brain MAChRs was more pronounced in aged than in young rats (50 and 25%, respectively), while that of ileal MAChRs was greater in young than in aged rats (50 and 35%). The normalization of cortical MAChRs was delayed in aged rats that of ileal MAChRs was delayed in young rats. As regards age-related changes of AChE and MAChRs in untreated rats, there was a 30% decrease of cortical and ileal AChE, no changes in Bmax of cortical MAChRs and a 45% deficit of ileal MAChRs. This was accompanied by only a little age-related decrease in sensitivity of the isolated ileum to cholinergic agonists. Additional experiments on the responsiveness of phosphatidyl inositol system stimulated with carbachol showed that accumulation of inositol phosphate both in cortical and ileum strip slices was higher in aged than in young rats. The overall data indicate that treatment- and age-related changes of AChR mechanisms in the ileum strip differ considerably from those in the brain. However, the increased efficiency of post-receptor mechanisms in old age is their common feature.

Age-related changes in acetylcholinesterase and its molecular forms in various brain areas of rats.

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 medulla-pons or the cerebellum.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of diisopropyl fluorophosphate on muscarinic-M1-receptors and on receptor-mediated responsiveness of the phosphatidyl-inositol system in the cerebral cortex of rats.

The effects of acute and repeated treatments with an anticholinesterase (anti-ChE) compound, diisopropylfluorophosphate (DFP) on M1-acetylcholine receptor (M1-AChRs) density and on M1-mediated breakdown of inositol phospholipids were studied in the cerebral cortex of rats. The DFP doses induced an about 75% inhibition of cortical ChE 48 hr after the last treatment. The acute treatment did not change Bmax of M1-AChRs (measured as 3H-pirenzepine binding), while 1-week and 2-weeks treatments induced their significant down-regulation, by 14 and 29%, respectively. The responsiveness of M1-AChRs was measured in cortical prisms as accumulation of inositol phosphate (IP) following stimulation with a cholinergic agonist, carbachol (from 10 to 1000 microM). The IP accumulation (expressed as ratio stimulated/basal IP content) was lower in acute DFP rats than in controls at few carbachol concentrations, and after 2 weeks at most carbachol concentrations. This resulted in a significant increase of EC50. The data indicate the involvement of cortical phosphatidyl inositol system during intoxication by anti-ChE agents, namely a decreased efficiency of post-receptor mechanisms.

Size and charge isomers of acetylcholinesterase in the cerebral cortex of young and aged rats.

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.

Adaptive processes of the central and autonomic cholinergic neurotransmitter system: age-related differences.

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.

In vivo and in vitro effects of diisopropyl fluorophosphate and paraoxon on individual molecular forms of rat brain acetylcholinesterase.

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.

Impaired recovery of brain muscarinic receptor sites following an adaptive down-regulation induced by repeated administration of diisopropyl fluorophosphate in aged rats.

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.