Lee J. Martin

Selective regional loss of exocytotic presynaptic vesicle proteins in Alzheimer’s disease brains

We tested whether regional or selective alterations in presynaptic proteins occur in Alzheimers disease (AD) and correlate with tests of cognitive function. We measured the levels of seven presynaptic proteins (synaptobrevin, synaptotagmin, SNAP-25, syntaxin, SV2, Rab3a, and synapsin I) by immunoblotting in postmortem tissue from four brain regions (hippocampus, entorhinal cortex, caudate nucleus, and occipital cortex). Three subject groups were studied: AD, possible/early AD (p-AD), and age-matched controls. Synaptobrevin and synaptotagmin were significantly reduced (29%, P<0.08; 38%, P<0. 07) in hippocampus in p-AD compared to controls. In definite AD compared to controls, selective regional reductions in vesicle proteins were found: synaptobrevin (46%, P<0.05), synaptotagmin (52%, P<0.01), and Rab3a (30%, P<0.05) in hippocampus; synaptobrevin (31%, P<0.01), synaptotagmin (15%, P<0.05), and Rab3a (44%, P<0.05) in entorhinal cortex. In contrast, the levels of two vesicle proteins (synapsin I and SV2) and two presynaptic membrane proteins (syntaxin and SNAP-25) were similar to controls. Synaptobrevin was the only vesicle protein reduced in AD in all four brain regions (occipital cortex 37%, P<0.05; caudate nucleus 31%, P<0.05). By univariate analysis of all cases, Mini-Mental State Examination, Blessed (BIMC) and Free Recall scores were strongly correlated with reduced levels of synaptic vesicle proteins synaptobrevin, synaptotagmin, and Rab3a in hippocampus and entorhinal cortex. These results suggest that there are selective and early defects in presynaptic vesicle proteins, but not synaptic plasma membrane proteins in AD and that defects correlate with cognitive dysfunction in this disease.

Protective effect of diazepam pretreatment on soman-induced brain lesion formation.

Histopathological analyses of brains of rats receiving a single 0.9 LD50 injection of soman, a potent anticholinesterase neurotoxin, revealed massive widespread lesions in the cerebral cortex and thalamus 4 weeks post-injection. Such lesions were not evidenced in rats receiving diazepam (2.2 mg/kg, i.m.) 10 min prior to soman treatment. Thus, anticonvulsant antidotes may aid in preventing extensive or permanent brain damage in rats surviving near-lethal soman dosages.

Scanning cytophotometric analysis of brain neuronal nuclear chromatin changes in acute T-2 toxin-treated rats.

Male Sprague-Dawley rats (200 g) were injected intraperitoneally with T-2 toxin, a trichothecene mycotoxin protein synthesis inhibitor, at dosages of 0.75, 1.0, 1.5, and 6.0 mg/kg (1 LD50 = 0.9 mg/kg) before decapitation at 8-hr postexposure. Correlative data were obtained on changes in physicochemical properties of nuclear chromatin, chromatin dispersion, and nuclear volume of cerebrocortical (layer III) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage, and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity, i.e., neuronal nuclear functioning. Conversely, increased F-DNA yield, chromophore area, and nuclear volume signify enhanced neuronal activation. At 8 hr following T-2 toxin exposure, cerebrocortical and striatal neurons exhibited a dose-dependent decrease in F-DNA hydrolyzability, i.e., impaired chromatin activity, and increases in both chromatin dispersion and nuclear volume. Microscopic observation revealed no gross evidence of T-2 induced neurotoxicity. These data indicate that T-2 toxin elicits both neurochemical injury and adaptive or compensatory processes simultaneously. The toxicological importance of observed nuclear alterations and the role of impairments in central nervous system metabolism in acute T-2 toxicity remain to be ascertained.

Quantitative cytophotometric analysis of brain neuronal RNA and protein changes in acute T-2 mycotoxin poisoned rats

Male Sprague-Dawley rats (200 g) injected intraperitoneally with T-2 toxin, a trichothecene mycotoxin protein synthesis inhibitor, at dosages of 0.75, 1.0, 1.5 and 6.0 mg/kg (1 LD50 = 0.9 mg/kg) were decapitated at 8 hr post-exposure. Data were obtained on changes in neuronal (perikaryal) RNA levels, protein contents and nucleolar volumes in cerebrocortical (layer III) and striatal (caudate-putamen) brain regions using quantitative azure B-RNA and Coomassie-protein cytophotometry and ocular filar micrometry. Correlative observations were made on changes in brain cytomorphology. Reductions in neuronal RNA/protein contents and nucleolar volume were used as indices of impaired perikaryal functioning. At 8 hr after T-2 toxin poisoning the following results were obtained in cerebrocortical and striatal brain compartments: neuronal RNA contents were generally maintained at control values in both brain regions, however, moderate RNA depletion was evidenced in the cerebral cortex with 1.5 mg/kg T-2 and in the striatum with a 6.0 mg/kg dose; neuronal protein levels were suppressed in a dose-dependent fashion within the cerebrocortex, while in the striatum there was no direct correspondence between protein loss and T-2 dosage; neuronal nucleolar volumes were typically maintained at control levels in both neuronal compartments. Microscopic observations revealed no gross evidence of T-2-induced brain cytopathology. These data indicate that T-2 toxin does not elicit direct cytopathic actions in these two brain regions, thus indicating that cerebrocortical and striatal compartments do not represent primary target sites of T-2 toxicant action.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain neuronal chromatin responses in acute soman intoxicated rats

Male Sprague-Dawley rats (200 g) were injected subcutaneously with soman, a potent neuronal acetylcholinesterase (AChE) inhibitor, at doses of 0.5, 0.8 and 1.0 LD50 (1 LD50=135 μg/kg) before decapitation at 1 and 24 h post-exposure. Correlative data were obtained on the severity of brain AChE inactivation and physicochemical changes in nuclear chromatin of cerebrocortical (layer V) and striatal neurons using Feulgen-DNA (F-DNA) cytophotometry and ocular filar micrometry. Decreased lability of neurons to F-DNA acid hydrolysis (reduced F-DNA yield), nuclear shrinkage and chromatin aggregation (decreased chromophore area) were used as indices of suppression of genomic template activity; conversely, increases in F-DNA yield and chromophore area signify enhanced neuroexcitation. At 1 hr post-soman there was a dose-dependent inactivation of AChE with a moderate increase in chromatin activation, i.e., nuclear hypertrophy and chromatin dispersion. At 24 hr post-soman there was a partial restoration of AChE activity, notably in striatal neurons, with a suppression in chromatin template activity. These data indicate that actions of soman on neuronal functioning are time-dependent. The absence of any dose-related neuronal chromatin changes may signify existence of non-cholinergic mediated events.

Effects of diazepam on soman-induced brain neuronal RNA depletion and lethality in rats

Studies were conducted to determine effects of the benzodiazepine anticonvulsant diazepam on soman induced brain neuronal RNA depletion and lethality in rats. Quantitative azure B-RNA cytophotometry was used to monitor RNA responses of cerebrocortical (layer V) and striatal neurons following dosages of 0.5, 0.9 and 1.5 LD50 soman (LD50 = 135 micrograms/kg, sc), whereas mean time of death and 24-h survival following 0.8, 1.2 and 1.5 LD50 were used to assess the antidotal efficacy of diazepam (2.2 mg/kg, im) pretreatment. Soman produced dose-dependent RNA depletion in both brain regions. This RNA impairment was almost completely prevented by diazepam, although neuronal RNA contents were generally slightly lower than corresponding control values. However, diazepam pretreatment was not associated with any change in mean time of death or in 24-h survival. The overall data suggest that excessive neural activity per se may underlie the genesis of soman-induced central metabolic impairments, but also appear to effectively dissociate epileptiform activity from lethal actions of soman.

Cytophotometric analyses of brain neuronal RNA in soman intoxicated rabbits

Quantitative azure B-RNA cytophotometry was used to monitor metabolic responses of individual neurons of the motor cortex (layer V) and caudate nucleus of soman (pinacolyl methylphosphonofluoridate) poisoned rabbits. Time- and dose-dependent RNA responses were related to the extent of acetylcholinesterase (AChE) inactivation of these 2 brain regions and to overt behavioral manifestations of toxication. A complex pattern of RNA responses was evidenced, with RNA depletion occurring at both arousal and convulsive doses of soman. In general, RNA changes paralleled previously reported metabolic responses observed in soman toxicated rats and mice: (1) linear dose-dependent suppression of RNA was evidenced during the depressant phase but not in the acute excitatory phase of toxication; and (2) RNA depletion was more severe following than during the appearance of excitatory symptoms. These data indicate that soman poisoning results in metabolic correlates of impaired rather than excessive CNS activation. It is postulated that metabolic disturbances are related to blockade of central cholinergic excitation, and that disruption of functionally integrated synaptic activity forms a critically important aspect of toxication.

Mesenteric mast cell degranulation in acute T-2 toxin poisoning

T-2 toxin-induced alterations in rat mesenteric mast cell granulation were measured by cytophotometric analyses of the metachromatic reaction of mast cell granules with azure B. Hypogranulation (diminution of metachromatic material) was observed 8 h following injections of T-2 toxin (0.5-1.5 LD50, i.p.). These data suggest that mast cell activation occurs during acute T-2 intoxication and raise the possibility that mast cell mediators may contribute to toxin-induced cardiovascular collapse.

Cytophotometric Analysis of Neuronal Chromatin and RNA Changes in Oxotremorine-Treated Rats

Abstract Neuronal nucleic acid responses were examined within the rat striatum and sensorimotor cortex (layer V) following single intraperitoneal injections of the central cholinergic-muscarinic agonist oxotremorine (0.1, 0.7, or 1.0 mg/kg). After stoichiometric Feulgen and azure B staining of brain sections, scanning-integrating microdensitometry was used to quantify Feulgen-deoxyribonucleic acid levels, changes in the susceptibility of chromatin to Feulgen acid hydrolysis (F-DNA yield) and azure B-ribonucleic acid (RNA) content of neurons on an individual basis. Changes in neuronal nuclear and nucleolar volumes were also determined histometrically. Within the striatum and sensorimotor cortex, oxotremorine produced marked dose-dependent elevations in both F-DNA yield and RNA content. These metabolic increases were typically paralleled by elevations in nuclear and nucleolar volumes. The data demonstrate that the oxotremorine-induced central muscarinic activation is associated with dose-related enhancements in neuronal chromatin template activity, RNA content, and protein synthetic capacity.

Effects of HI-6 and pralidoxime on neuronal RNA in thalamic cholinergic sites ☆

Quantitative azure B-RNA cytophotometry was employed to compare effects of the oximes HI-6 and pralidoxime (2-PAM) to those of atropine sulfate (AS) on neuronal RNA metabolism in the thalamic ventrobasal nuclear complex (VBC) and nucleus reticularis (NR). The ability of these compounds to mitigate soman (pinacolyl methylphosphonofluoridate)-induced neuronal RNA alterations (i.e., VBC-RNA depletion/NR-RNA elevation) in these muscarinic cholinergic sites was also determined. Generally, HI-6 (125 mg/kg, i.p.) and 2-PAM (43.2 mg/kg, i.m.) elicited similar patterns of neuronal RNA changes, i.e., diminution of VBC-RNA and NR-RNA with oximes alone; partial amelioration of soman (1.5 LD50, s.c.)-induced VBC-RNA loss; and slight or no effect on soman induced NR-RNA accumulation. HI-6 produced more severe RNA reduction than 2-PAM in both brain regions of non-poisoned rats, whereas 2-PAM was more effective in reversing the effects of soman in these two regions. The muscarinic antagonist, AS, also produced VBC-RNA depletion and partially counteracted the VBC-RNA loss in soman intoxicated rats. Unlike the oximes, however, AS resulted in NR-RNA accumulation and it also antagonized soman induced NR-RNA elevation. Neither oxime reactivated soman inhibited brain acetylcholinesterase but HI-6 did reactivate appreciable plasma cholinesterase. The overall data suggest that HI-6 and 2-PAM do exert pharmacologic actions on cholinergic neurons in the rat CNS. However, the greater effectiveness of HI-6 over 2-PAM in countering lethal actions of soman does not appear to be correlated with oxime mediated restoration of neuronal RNA levels in these two cholinergic regions.(ABSTRACT TRUNCATED AT 250 WORDS)