Chick embryo exposure to carbamates alters neurochemical parameters and behavior.

Recent evidence has shown that exposure to pesticides can lead to long-term neurophysiological and functional deficits. We have demonstrated previously that locomotion in chicks exposed to some organophosphates and carbamates could be altered persistently without concomitant central or peripheral esterase inhibition. Furthermore, histopathology of the ataxic chickens showed no lesions in either the central or peripheral systems. In this study, we examined whether locomotion alterations seen in chicks exposed in ovo to carbaryl and aldicarb are accompanied by perturbations in particular central neurotransmitter systems. Carbaryl and aldicarb were injected in ovo on day 15 of incubation at 6, 16 and 65 mg kg-1 egg weight and at 0.2, 0.4 and 35 mg kg-1 egg weight, respectively. Neurotransmitter levels (assayed by high performance liquid chromatography with electrochemical detection) and locomotion were measured at various times (1-43 days) after dosing. At the lower doses of both carbaryl and aldicarb, a trend towards prolonged decreases in cerebral dopamine and homovanillic acid was seen. The high dose of carbaryl significantly reduced dopamine and the high dose of both compounds significantly decreased homovanillic acid and 5-hydroxyindoleacetic acid. Persistent locomotion alterations were observed only at the higher doses of both carbaryl and aldicarb when the specific neurotransmitters measured returned to normal levels.

Time course of serotonergic afferent plasticity within rat spinal trigeminal nucleus following infraorbital nerve transection.

High-performance liquid chromatography with electrochemical detection (HPLC-ED) and immunocytochemistry were used to examine the time course of serotonergic afferent plasticity within trigeminal subnucleus interpolaris (SpVi) following infraorbital nerve (ION) transection in adult rats. Biochemical analysis was also performed in trigeminal subnucleus caudalis (SpVc) to examine the possibility of transient lesion-induced changes in this region. No significant changes in serotonin (5-HT) or 5-hydroxyindoleacetic acid (5-HIAA) concentration, or in density of 5-HT-immunoreactive (5-HTIR) axonal varicosities were observed in either subnucleus on the lesioned side, up to 51 days following ION cut. However, at 76-79 days post-lesion, a significant increase in 5-HT concentration was again demonstrated within SpVi.

Biochemical and anatomical consequences of adult infraorbital nerve transection for serotonergic afferents within rat trigeminal subnuclei interpolaris and caudalis.

Immunocytochemistry and high-performance liquid chromatography with electrochemical detection (HPLC-ED) were used, more than 76 days after infraorbital nerve (ION) transection, to examine the distribution and density of serotonin-immunoreactive (5-HTIR) axons, as well as serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) content, within the infraorbital (IO) regions of subnuclei caudalis (SpVc) and interpolaris (SpVi). In SpVi, increases in 5-HT concentration and in density of 5-HTIR axonal varicosities were observed on the lesioned side. No changes were seen in SpVc.

Biochemical and behavioral effects of intrahippocampal AF64A in rats.

AF64A (ethylcholine aziridinium, 1 nmole) injected into the dorsal hippocampus of the rat decreased choline acetyltransferase activity there by 20% without greatly affecting adjacent areas. The decrease was maximal by 3 days, and persisted for at least 3 weeks. The acetylcholine concentration at the injection site was decreased by 25-30% from 3 days to 4 weeks. Rats were trained on a continuous reinforcement (CRF) food-reinforced lever press schedule and then injected bilaterally in the dorsal and ventral hippocampus. Subsequent switching to a daily CRF-extinction schedule resulted in increased responding during extinction compared to controls which persisted for at least 13 session. However, injection after switching schedules increased it for only 2 sessions. This indicates that the persistently increased extinction responding is due mainly to impaired learned habituation to a new schedule. Most of the extinction effect of the intrahippocampal AF64A was due to its injection at the dorsal site. Separate rats which were trained on the 8-arm radial maze task (a test of short-term spatial working memory) and injected as above only showed marginally impaired task performance even at higher doses. We conclude that even relatively minor, localized, cholinergic deficits confined to the hippocampus can produce significant learning and memory impairments in situations where intermediate or long term memory formation is required.

Computer program for the parametric and nonparametric comparison of several groups to a control.

A BASIC program is described which, upon the input of raw data from an experiment comparing several treatment groups to a control, will output group parameters (mean, SEM), test for outliers in each group (maximum normalized residual test), and examine the homogeneity of variance (Bartlett's test). Subsequently, one-way ANOVA and Dunnett's multiple comparison test are performed. The user also has the option of examining the data by nonparametric means using the Kruskal-Wallis test and a nonparametric multiple comparison test (Dunn's test). As a convenience, the program contains algorithms to generate critical table values for all the tests employed.

Ornithine as a precursor of neurotransmitter glutamate: effect of canaline on ornithine aminotransferase activity and glutamate content in the septum of rat brain.

Local injections of L-canaline into the septum produce a rapid and almost complete inhibition of ornithine aminotransferase activity followed by a decrease in glutamate content in this region. The time-course of canaline action shows the existence of two glutamate pools with different sizes and half-life values. Surgical lesions of the hippocampal-septal glutamatergic pathway affected the site and kinetics of the small pool of glutamate in the septum, suggesting the participation of ornithine aminotransferase in the synthesis of this pool. This indicates a possible role of ornithine as a precursor of the transmitter glutamate. The localization of ornithine aminotransferase does not seem, however, to be specific for the nerve-terminal compartment. The data obtained allow estimation of the turnover rate of the specific pool of neurotransmitter glutamate.

GABAergic control of the cholinergic projections to the frontal cortex is not tonic.

The turnover rate of acetylcholine was measured in the frontal cortex of rats after either microinjection of bicuculline into the substantia inominata (the source of the cortical cholinergic innervation) or kainic acid lesioning of the nucleus accumbens (the source of GABAergic innervation of the substantia inominata). Neither treatment affected cortical acetylcholine metabolism, suggesting that the GABAergic inhibition of the substantia inominata-cortical cholinergic pathway is not tonic.

Behavioral and neurochemical differentiation of specific projections in the septal-hippocampal cholinergic pathway of the rat.

In the rat, an intraseptal injection of the gamma-aminobutyric acid (GABA) agonist muscimol decreases the turnover rate of acetylcholine in the hippocampus and, during extinction of a food-reinforced lever-press response, increases extinction responding in a dose-dependent manner. Intraseptal beta-endorphin decreases the turnover rate of hippocampal acetylcholine through activation of septal GABAergic interneurons and increases extinction responding. On the other hand, intraseptal substance P, which decreases the turnover rate of hippocampal acetylcholine in a manner unrelated to septal GABAergic mechanisms, fails to increase extinction responding. The turnover rate of acetylcholine in various hippocampal regions after intraseptal injection of muscimol and substance P was also studied. Muscimol decreases the acetylcholine turnover rate only in the ventral hippocampus, whereas substance P decreases it only in the dorsal hippocampus. We hypothesize that a lowering in the cholinergic input to the ventral hippocampus is capable of increasing extinction responding, whereas a decrease in the input to the dorsal hippocampus is without such an effect. Hence, the cholinergic projections to the two hippocampal areas are modulated by different transmitter systems and have different physiological functions.

Simultaneous modulation of hippocampal cholinergic activity and extinction by intraseptal muscimol.

The relationship between regulation of acetylcholine metabolism in the septal-hippocampal pathway and extinction of a food reinforced lever press response was investigated by comparing the turnover rate of acetylcholine (TRACh) in the rat hippocampus with the amount of responding during extinction after intraseptal injection of the gamma-aminobutyric acid receptor agonist muscimol. Doses (0.3-3.0 nmol) which decreased the TRACh in the hippocampus also increased the responding during extinction over that of saline controls. Responding during the continuous reinforcement schedule before extinction was also increased, but to a lesser extent. Higher doses (10-30 nmol) further decreased the TRACh in the hippocampus, decreased it in the cortex and were accompanied by irregular responding and sedation. The TRACh in the hippocampus was also measured in drug-free rats undergoing extinction after training on a continuous reinforcement or variable interval 60 sec reinforcement schedule. Although the variable interval 60 sec reinforcement schedule rats responded more than the continuous reinforcement rats during extinction, there were no differences between the TRACh in the hippocampus. The present results indicate that the decrease in the hippocampal TRACh which is produced by intraseptal muscimol is accompanied by an increase in the response rate during extinction, but that operantly induced differences in this behavior are not accompanied by detectable changes in hippocampal TRACh.

beta-Carbolines enhance shock-induced suppression of drinking in rats.

By using Vogel's method to test the anxiolytic action of benzodiazepines and reducing the intensity of the current delivered to the drinking tube, it is possible to distinguish the pharmacological activity of three types of ligands for the benzodiazepine recognition site. An anticonflict action typical of anxiolytic benzodiazepines, a proconflict action typical of many beta-carbolines, including FG 7142 (beta-carboline-3-carboxylic acid ethyl ester methyl amide), and an antagonistic action of the proconflict and anticonflict actions typical of RO 15-1788 (ethyl-8-fluoro-5, 6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-alpha]-[1, 4]-benzodiazepine-3-carboxylate) and CGS 8216 (2-phenylpyrazolo[4,3-c]quinolin-3-(5H)-one). Pentylenetetrazole, which causes convulsions by interacting with a subunit of the gamma-aminobutyric acid receptor that is different from the benzodiazepine recognition site, also induces a proconflict action that is antagonized by anxiolytic benzodiazepines but not by RO 15-1788.

Interstrain comparison of avoidance behavior and neurochemical parameters of brain cholinergic function.

Five rat strains (Long-Evans Hooded, Zivic Miller, Lewis, Buffalo and Fischer-344) were tested in a shuttlebox conditioned avoidance task and the differences in the performance levels among the strains were noted. In parallel experiments using naive rats, the acetylcholine concentrations in eight brain regions and the acetylcholine turnover rate in five brain regions were determined for these strains. Interstrain differences in these parameters were found but no correlation between avoidance performance and either of these measures was apparent in any brain region studied. In separate experiments, no differences were found in the hippocampal acetylcholine concentration or the turnover rate among good performing Hooded, poor performing Hooded or untested Hooded rats. Similarly, no differences in regional acetylcholine turnover rates were found between naive rats of the Iowa Reactive and Nonreactive strains. [3H]-QNB (quinuclidinyl benzilate) binding was studied in three brain regions in the five strains, but no large interstrain differences in binding characteristics were found. In contrast to interpretations of other workers based on less direct assay methods involving fewer strains, we conclude that no strong correlation exists between avoidance performance ability and basal levels of brain cholinergic activity.

Differences in the kinetics of axonal transport for individual lipid classes in rat sciatic nerve.

Lipid precursors ([2-3H]glycerol for phospholipids and [3H]acetate for cholesterol) were injected into the L-5 dorsal root ganglion of adult rats. At various times, animals were killed, the ganglion and consecutive 5-mm segments of sciatic nerve were dissected, and lipids were extracted and analyzed by TLC. Individual lipid classes exhibited markedly different transport patterns. The crest of radioactive phosphatidylcholine moved as a sharply defined front at about 300 mm/day, with a relatively flat plateau behind the moving crest. Although some radioactive phosphatidylethanolamine also moved at the same rate, the crest was continually attenuated as it moved so that a gradient of radioactive phosphatidylethanolamine along the axon was maintained for several days. Transported diphosphatidylglycerol exhibited a defined crest, as did phosphatidylcholine, but moved at about half the rate. Labeled cholesterol was transported at a rapid rate similar to that for phosphatidylcholine and phosphatidylethanolamine, but like phosphatidylethanolamine, the initial moving crest of radioactivity was continually attenuated. Relative to the phospholipids, cholesterol showed a more prolonged period of accumulation in the axons and was more metabolically stable. We propose that most labeled phosphatidylcholine, phosphatidylethanolamine, and cholesterol is transported in similar (or the same) rapidly moving membranous particles. Once incorporated into these particles, molecules of phosphatidylcholine tend to maintain associated with them during transport. In contrast, molecules of phosphatidylethanolamine and cholesterol in these transported particles exchange extensively with unlabeled molecules in stationary axonal structures. Diphosphatidylglycerol, localized in a specialized organelle, the mitochondrion, is transported at a slower rate than other phospholipids, and does not exchange with other structures.

Inhibition of acetylcholine turnover rate in rat hippocampus and cortex by intraventricular injection of adenosine analogs.

The effects of i.c.v. administration of adenosine receptor agonists and antagonists on the turnover rate of acetylcholine (TRACh) in various areas of the rat brain were examined in an effort to better understand the role of adenosine as a neuromodulator or cotransmittr. TRACh was determined by gas chromatographic-mass fragmentographic analysis of the rate of deuterium incorporation into ACh and choline during a constant rate infusion of deuterated phosphorylcholine. The i.c.v. administration of the adenosine receptor agonist, 2-chloroadenosine (2-CIAdo), in a dose of 82 nmol failed to change the ACh or choline content of any of the brain areas examined. This dose of 2-CIAdo elicited significant reductions in the TRACh in both the hippocampus and frontal cortex, but not in the striatum. The extent of the TRACh reduction was 67 and 36% in hippocampus and cortex, respectively. This inhibition of TRACh elicited by 2-CIAdo was antagonized by pretreatment (i.c.v.) with theophylline (278 nmol), suggesting that an activation of adenosine receptors is operative in the action of 2-CIAdo. Further support for a participation of adenosine receptors in the action of 2-CIAdo was obtained by comparing the effects of the L- and D-isomers of phenylisopropyladenosine (PIA) on TRACh. The i.c.v. administration of L-PIA (65 nmol) elicited a 79% reduction in the TRACh in the hippocampus, whereas D-PIA (65 nmol i.c.v.) had no significant effect on hippocampal TRACh. This finding supports the view that these effects on TRACh may be mediated by adenosine A1 receptors, but not by A2 receptors, because the former, but not the latter, display marked stereoselectivity toward PIA. It also was demonstrated that intraseptal injections of L-PIA or theophylline failed to reduce the TRACh in the hippocampus, suggesting that adenosine receptors located in the septum are not operative in mediating the i.c.v. action of PIA.

Axonal transport of the mitochondria-specific lipid, diphosphatidylglycerol, in the rat visual system.

Rats 24 d old were injected intraocularly with [2-3H]glycerol and [35S]methionine and killed 1 h-60 d later. 35S label in protein and 3H label in total phospholipid and a mitochondria-specific lipid, diphosphatidylglycerol(DPG), were determined in optic pathway structures (retinas, optic nerves, optic tracts, lateral geniculate bodies, and superior colliculi). Incorporation of label into retinal protein and phospholipid was nearly maximal 1 h postinjection, after which the label appeared in successive optic pathway structures. Based on the time difference between the arrival of label in the optic tract and superior colliculus, it was calculated that protein and phospholipid were transported at a rate of about 400 mm/d, and DPG at about half this rate. Transported labeled phospholipid and DPG, which initially comprised 3-5% of the lipid label, continued to accumulate in the visual structures for 6-8 d postinjection. The distribution of transported material among the optic pathway structures as a function of time differed markedly for different labeled macromolecules. Rapidly transported proteins distributed preferentially to the nerve endings (superior colliculus and lateral geniculate). Total phospholipid quickly established a pattern of comparable labeling of axon (optic nerve and tract) and nerve endings. In contrast, the distribution of transported labeled DPG gradually shifted toward the nerve ending and stabilized by 2-4 d. A model is proposed in which apparent "transport" of mitochondria is actually the result of random bidirectional saltatory movements of individual mitochondria which equilibrate them among cell body, axon, and nerve ending pools.

Effect of triethyl tin on myelination in the developing rat.

Myelinogenesis in developing rats was studied following chronic dosing with triethyl tin (TET), at a level of 1.0 mg TET/kg body wt/day. Experiments included starved controls with body weights depressed by 17 to 40% to equal those of the TET-treated groups. Rats at ages of 16, 21, and 30 days showed decreases relative to well-nourished controls in body weight, forebrain weight, myelin yield, cerebroside level, and specific activity of brain 2',3'-cyclic nucleotide-3'-phosphohydrolase when dosed with TET. At 30 days, myelin and cerebroside yields were reduced by approximately 55%, while CNP activity was reduced by less than 20%. No differences in the forebrain myelin protein composition between control, starved, and TET animals were noted. The rate of myelin protein synthesis relative to brain total protein (assayed by incorporation of intracranially injected [3H]glycine into brain homogenate and myelin proteins) was decreased in the TET rats in proportion to the decreased yield of myelin, but no particular myelin protein was preferentially affected. Matching starved controls exhibited similar body weight decreases, less pronounced forebrain weight decreases, and little or no decrease in myelin concentration. There was a relative increase in the myelin protein synthesis rate in the starved rats, indicating preferential utilization of limited protein precursors for myelin protein synthesis. Spinal cord myelin was also decreased in the TET rats, but less severely than in the forebrain. At all ages optic, but not sciatic, nerves showed decreases in myelin concentration with TET treatment. We conclude that TET inhibits forebrain growth and CNS myelination more severely than can be accounted for by a general metabolic insult.

Cholesterol is a component of the rapid phase of axonal transport.

Twenty-two-day-old rats were injected intraocularly with [3H]acetate and killed between 1 hr and 35 days later. Cholesterol was isolated from the retinas, optic tracts, lateral geniculate bodies, and superior colliculi. Within the retina, radioactivity was rapidly incorporated into cholesterol with maximal labeling present one hour after injection. Transported labeled cholesterol (contralaterally corrected for systemic background labeling) was present in the superior colliculus by three hours. Radioactive cholesterol accumulated in all visual structures throughout the 35-day period, but the rate of accumulation was maximal at about the time of arrival of the initial pulse of radioactivity. Colchicine treatment of the retina blocked transport of cholesterol but not its synthesis by the retina. The results indicate that cholesterol is rapidly transported in the visual system and also released from the retina for a prolonged period after its synthesis.

The effect of hibernation on the positional distribution of ethanolamineglycerophospholipid fatty acids in hamster brain membranes.

Microsomal and myelin membrane fractions were prepared from the brains of warm-adapted (room temperature) and hibernating Syrian Hamsters (Mesocricetus auratus). Ethanolamineglycerophospholipids were isolated and subjected to a fractionation scheme to separate the fatty acids of the plasmenylethanolamine and the phosphatidylethanolamine 1 and 2 positions. The major changes in microsomal phosphatidylethanolamine with hibernation were relative increases in 18:1 at the 1 position and 20:4(n-6) in the 2 position. In myelin, 18:1 increased and 18:0 decreased at the 1 position while the 2 position showed an increase in 16:0 and a decrease in 22:6(n-3). Plasmenylethanolamine fatty acids also changed in microsomes and myelin although the magnitudes were not as great and confined to longer chain fatty acids. In both membranes, fatty acid alterations were position-specific, and no new types of fatty acids were introduced at any position.