Deficit in selective and divided attention associated with cholinergic basal forebrain immunotoxic lesion produced by 192-saporin; motoric/sensory deficit associated with Purkinje cell immunotoxic lesion produced by OX7-saporin.

The immunotoxin 192-saporin, infused intracerebroventricularly into rats, destroys cholinergic neurons in the basal forebrain nuclei. Doses required for complete cholinergic loss also kill some Purkinje cells. The immunotoxin OX7-saporin, when infused intraventricularly into rats, destroys Purkinje cells in a pattern similar to that produced by 192-saporin, without affecting cholinergic neurons. Thus, we used OX7-saporin to distinguish behavioral effects of 192-saporin due to cerebellar damage versus those due to cholinergic cell loss. Three doses of 192-saporin (1.6, 2.6, and 3.3 micrograms/rat) were chosen along with a dose of OX7-saporin (2.0 micrograms/rat) that produced Purkinje loss equivalent to the two highest doses of 192-saporin. Groups of Fischer-344 rats were trained in the multiple choice reaction time task and retested with more complex tasks after lesioning. They were also tested in the water maze, passive avoidance, acoustic startle, and open field. The OX7-saporin group exhibited changes in many tests suggesting hypermotility and sensory deficits. The 192-saporin groups differed from the OX7-saporin group when they displayed deficits in multiple choice reaction time tasks in which novel challenges were introduced, including sessions with a noise distractor, shortened and lengthened intertrial intervals, and use of nine instead of five sources of light stimulus. The 192-saporin groups showed no impairment in the other tasks. The cholinergic basal forebrain lesion may mask some of the effects of cerebellar damage up to a threshold after which effects of Purkinje cell loss predominate when 192-saporin is administered intraventricularly.

192 immunoglobulin G-saporin produces graded behavioral and biochemical changes accompanying the loss of cholinergic neurons of the basal forebrain and cerebellar Purkinje cells.

Immunolesions of the cholinergic basal forebrain were produced in rats using various intraventricular doses of the immunotoxin 192 immunoglobulin G-saporin: 0.34, 1.34, 2.0, 2.7 and 4.0 micrograms/rat. A battery of behavioral tests, chosen on the basis of reported sensitivity to conventional medial septal or nucleus basalis lesions, was administered. Dose-dependent impairments were found in acquisition, spatial acuity and working memory in the water maze. Dose-dependent hyperactivity in the open field and in swimming speed was observed. The highest dose group (4.0 micrograms) exhibited motoric disturbances which were particularly apparent in swimming and in clinging to an inclined screen. Response and habituation to acoustic startle were diminished in the three higher dose groups. Histological results from acetylcholinesterase and low-affinity nerve growth factor receptor staining showed that the lesion was selective for cholinergic neurons bearing p75 nerve growth factor receptors in the basal forebrain nuclei. However, some Purkinje cells in the superficial layers of the cerebellum were also destroyed at the higher doses of immunotoxin. The activity of choline acetyltransferase, used as a marker of cholinergic deafferentation in regions innervated by the basal forebrain nuclei, was decreased with increasing doses to a plateau level of about 90% (average depletion) for the two highest dose groups. These two groups were the only ones to exhibit consistent and severe behavioral impairments on all behavioral tests performed. Thus, for a relatively selective cholinergic basal forebrain lesion, almost a 90% reduction in choline acetyltransferase activity is needed to produce substantial behavioral deficits. It appears that either a considerable safety factor exists or robust compensatory mechanisms can ameliorate behavioral deficits from a major, but incomplete loss of cholinergic basal forebrain innervation.

Behavioral and biochemical consequences of combined lesions of the medial septum/diagonal band and nucleus basalis in the rat when ibotenic acid, quisqualic acid, and AMPA are used.

Combined lesions in the medial septum/diagonal band and nucleus basalis magnocellularis (NBM) in rats were produced using three excitotoxins, ibotenate (Ibo), quisqualate (Quis), and AMPA. Reductions in choline acetyltransferase (ChAT) activity differed in the cortical regions for the three toxins (AMPA > Quis > Ibo), but were fairly similar in the hippocampus. ChAT activities were not reduced in the globus pallidus, but AMPA reduced ChAT in the amygdala. Lesions with all three toxins produced similar decrements in hippocampal and posterior cortical serotonin levels. A small reduction in posterior cortical norepinephrine was detected for Quis and Ibo lesions. Spatial memory impairments were found for all three toxin groups compared with controls in acquisition, platform reversal, and a spatial probe in the water maze. The learning deficit was greatest with the Quis lesion and equivalent for the Ibo and AMPA lesions. There was no deficit in single trial passive avoidance retention for the Ibo and AMPA groups. The AMPA group was slower than controls on both training and retention trials to enter the dark compartment. This group also showed a tendency to hypoactivity as measured in an open-field test. Excitotoxic infusions into medial septum/diagonal band and NBM produced spatial mnemonic deficits which do not parallel reductions in overall ChAT activity and do not resemble the profile of behavioral changes previously reported for NBM lesions alone using these toxins.

Time course of cholinergic and monoaminergic changes in rat brain after immunolesioning with 192 IgG-saporin.

192 IgG-saporin, an immunotoxin targeted at the low affinity NGF receptor, was infused into the lateral ventricle of rat brain. Three days and one week post lesion, choline acetyltransferase activity was markedly decreased in cortex, hippocampus, olfactory bulbs, and septum (brain regions innervated by the cholinergic neurons of the basal forebrain) with no change in cerebellum, striatum or pons. Measurement of monoamine levels revealed increases in HVA, DOPAC and dopamine, primarily in the olfactory bulbs at the 28-day time point only, suggesting a compensation for cholinergic inactivity. High levels of basal forebrain cholinergic lesioning can be obtained with this immunotoxin with minimal or no effects on monoaminergic or other cholinergic systems.

Changes in CSF neurotransmitters in infantile spasms.

Cerebrospinal fluid (CSF) from 7 patients with infantile spasms (mean age: 6.7 months) was collected before and after treatment with adrenocorticotropic hormone (ACTH). The concentration of neurotransmitter metabolites was analyzed using high-performance liquid chromatography and compared to the metabolite concentration in the CSF from 7 age-matched controls (mean age: 6.1 months). Pretreatment levels of CSF 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid, 3-methoxy-4-hydroxyphenyl glycol (MHPG), and kynurenine were significantly lower in infantile spasm patients compared to controls. Following treatment, marked increases in 5-HIAA and decreases in kynurenine levels were observed in the CSF of the 5 infants whose seizures were eliminated or reduced by ACTH. In the 2 nonresponders 5-HIAA levels decreased. The level of MHPG was reduced slightly in 5 infants, including the 2 nonresponders, and was increased in 2 responders. CSF homovanillic acid levels increased in 4 infantile spasm infants and decreased in 3 following ACTH. These data demonstrate that the presence of seizures in infantile spasms is associated with a significant decrease in serotonergic activity and that elimination of seizures by ACTH is accompanied by increased serotonin turnover. The simultaneous increase of 5-HIAA and decrease of kynurenine, an alternate metabolite of tryptophan, suggests an underlying disturbance of tryptophan metabolism in infantile spasms. The possibility that elimination of seizures by ACTH may be related to decreased production of certain kynurenine metabolites, particularly quinolinic acid, is discussed.

Correlation between plasma physostigmine concentrations and percentage of acetylcholinesterase inhibition over time after controlled release of physostigmine in volunteer subjects.

Five to six subjects ingested doses of controlled-release physostigmine salicylate tablets (9, 12, and 15 mg) followed by sequential blood drawing for measurement of plasma physostigmine concentrations and percentage of acetylcholinesterase (AChE) inhibition. Both plasma physostigmine concentrations and percentage of AChE inhibition demonstrated dose proportionality to three doses of ingested drug. Plasma physostigmine concentrations correlated with percentage of AChE inhibition across time by dose and across all doses and subjects tested. These data should be helpful to physicians in adjusting physostigmine dosing, enabling them to use the relatively simple and widely available AChE assay to approximate plasma physostigmine concentrations.

Sensitive analysis of plasma physostigmine levels using dual-cell electrochemistry in the redox mode.

A column liquid chromatographic method using dual-electrode, redox electrochemical detection has been developed for measuring plasma and cerebrospinal fluid physostigmine levels. The method is suitable for detecting drug levels in a geriatric population following oral ingestion of sustained-release physostigmine preparations and for determining the pharmacokinetics of these preparations in biological fluids.

Substrate specificity and partial characterization of the PAF-acylhydrolase in human serum that rapidly inactivates platelet-activating factor.

The structure of the potent inflammatory mediator, platelet-activating factor, is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, PAF-acether). Human sera contain an acid labile factor (ALF) that is a Ca+2-independent 2-acylhydrolase-specific for AGEPC and AGEPC-like molecules. The enzyme functions by catalytically removing the sn-2 acetyl moiety from AGEPC, producing the biologically inactive sn-2 hydroxy form or 2-lyso-GEPC. Incubation of ALF with sn-2 acyl PAF analogs indicated that the enzyme hydrolyzes the sn-2 fatty acid only if the chain length is five carbons or less, the sn-1 position fatty acid length is greater than 10 carbon units, and at least one methyl group is present on the terminal amine of the choline group. The enzyme was active with either an ether or ester linkage at the sn-1 position. ALF is inactivated by heating to 65 degrees C for 30 min. It is pronase and trypsin sensitive but resistant to papain and papain with dithiothreitol. Further characteristics of human ALF indicated a broad pH range of activity with an optimum of pH 6.2 and an isoelectric point of 6.2 to 6.7. The specificity and Ca+2 independence of human ALF sets it apart from phospholipase A2. It is proposed that human ALF be called human serum PAF-acylhydrolase to distinguish it from other hydrolases currently known to exist.

Human serum acid-labile factor is an acylhydrolase that inactivates platelet-activating factor.

Platelet-activating factor (PAF) is a phospholipid that activates platelets, induces inflammation, and causes profound alteration in the cardiopulmonary system. PAF from rabbit basophils and hog leukocytes is 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC). Human and other mammalian serums contain an acid-labile factor (ALF) that rapidly inactivates AGEPC. ALF is associated with low-density lipoproteins but can be dissociated from the lipoproteins with detergent followed by ultracentrifugation. Delipidated ALF has an isoelectric point of approximately 7.1, its molecular weight is unknown, and it will not react with goat anti-whole human serum, antialbumin, anti-alpha- or anti-beta-lipoproteins, or antiapolipoproteins A or B. ALF has the following characteristics: 1) is acid labile; 2) is Ca2+ independent; 3) has a pH optimum of 6.2; 4) can hydrolyze a four-carbon but not a six-carbon or longer chain fatty acid at the sn-2 position; 5) is independent of an ester or ether linkage at the sn-1 position; 6) is incapable of hydrolyzing sn-2-acetylphosphatidylethanolamine, which indicates the need for at least one methyl group on the choline moiety of AGEPC; 7) requires between 5 and 16 carbons at the sn-1 position to remove a three- or four-carbon fatty acid on the sn-2 position; 8) is inactivated by heating to 65 C for 30 min; 9) is pronase and trypsin sensitive but papain resistant; and 10) is a hydrophobic molecule and thus behaves like a membrane-associated enzyme. Thus, ALF is a specific phosphatide 2-acylhydrolase.

The presence of platelet-activating factor (PAF) in normal human mixed saliva.

This report describes PAF activity in normal human mixed saliva from each of 24 randomly selected donors. The human salivary PAF (HS-PAF) was similar to rabbit basophil PAF (acetyl glyceryl ether phosphorylcholine or AGEPC) with respect to the following characteristics: 1. HS-PAF co-chromatographed with the standard rabbit basophil AGEPC and with synthetic AGEPC. 2. HS-PAF and AGEPC were unaffected by hirudin, indomethacin, and creatine phosphate/creatine phosphokinase, the respective inhibitors of platelet activation induced by thrombin, arachidonic acid, and ADP. 3. HS-PAF and AGEPC were inactivated by a 5-min incubation with ALF, the acid-labile factor in normal human serum that rapidly destroys AGEPC. 4. HS-PAF was demonstrated to be functionally similar to AGEPC by cross-desensitization experiments. In the absence of Ca++, HS-PAF and AGEPC cross-desensitized washed rabbit platelets to subsequent stimulation by either HS-PAF or AGEPC after recalcification. 5. HS-PAF was demonstrated to be structurally similar to AGEPC by several simple chemical tests for functional groups.

Abnormal immunoglobulins in severe combined immunodeficiency: analysis by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis.

The plasma immunoglobulins of patients with severe combined immunodeficiency were studied by immunoelectrophoresis and, following isolation by affinity chromatography, by SDS-polyacrylamide gel electrophoresis. Immunoglobulins in plasma from the eight patients studied were immunoelectrophoretically abnormal. Although certain of the immunoglobulins in plasma from five patients could not be identified antigenically, all possessed two mu determinant-bearing proteins with abnormally fast electrophoretic mobilities. Molecular analysis of immunoglobulins of three of these patients revealed two mu heavy chains of abnormally low molecular weight which lacked the ability to polymerize into the pentameric structure of IgM. The failure of concanavalin A to precipitate these molecules suggests that they lack the carbohydrate moiety of normal IgM. Using these techniques, we documented the acquisition of normal IgM synthesis by a patient grafted with maternal leukocytes and the partial immunologic development of a child maintained under gnotobiotic conditions. In the latter patient, between the age of 1 and 4 years, an abnormal mu component disappeared from plasma and normal IgM appeared.