Ruth A. Booth

Simultaneous measurement of endogenous and deuterium-labeled tracer variants of choline and acetylcholine in subpicomole quantities by gas chromatography-mass spectrometry.

Abstract A method is described for the simultaneous analysis of endogenous and d 4 -labeled tracer variants of choline and acetylcholine by a gas chromatograph/mass spectrometer system operated in the specific ion detection mode, using d 9 -labeled variants of both choline and acetylcholine as internal standards. The detection limit is approximately 10 −13 moles of both tracer and endogenous variants.

Acetylcholine turnover estimation in brain by gas chromatography/mass spectrometry

Abstract A gas chromatograph/quadrupole mass spectrometer system has been employed to estimate the turnover of acetylcholine in mouse brain in vivo following a pulse intravenous injection of choline, using discrete deuterium labelled variants of choline and acetylcholine as tracer and internal standards. There appear to be two functional pools with turnover rates of 1.4 and 7.9 nmol gm −1 min −1 .

Mechanisms of tolerance to the anticholinesterase, DFP: Acetylcholine levels and dynamics in the rat brain

Abstract The effect of diisopropylfluorophosphate (DFP) on total and regional brain acetylcholine (ACh) levels and dynamics was investigated in rats as they developed tolerance to DFP. Microwave irradiation and integrated gas chromatography mass spectrometry (GCMS) were used in all assays. Significant increases in total brain ACh was seen at 4 through to 48 hr after an acute injection of DFP and after 1 through 22 days of chronic administration of DFP. Total brain choline levels were significantly decreased at 4 through 24 hr after DFP administration. Choline levels then became significantly greater than control values after 10 and 22 days of chronic treatment with DFP. No changes were seen in ACh synthesis or choline uptake. Regional brain levels of ACh or choline were not significantly changed 24 hr after acute administration of DFP. These results indicate that behavioral tolerance to DFP is not due to end-product inhibition of ACh synthesis. It is concluded that the probable mechanisms of tolerance may involve changes in presynaptic muscarinic receptors, as well as the changes in postsynaptic receptors which have already been verified.

Localized in vivo 1H magnetic resonance spectroscopy and in vitro analyses of heterogeneous brain tumors.

Results of magnetic resonance spectroscopic (MRS) studies of the chemical patterns in brain tumors have been mconsistent. Actual biochemical correlations are needed. In 2 patients with heterogeneous intracranial tumors, in vivo 1H MRS and in vitro biochemical analyses were correlated. Histology confirmed the tumor heterogeneity. Choline was elevated in the cellular portion of both tumors but decreased in the necrotic or cystic portions. Creatine was diffusely decreased while lactate was elevated in all regions of both tumors. Furthermore, the increase in the choline peak on 1H MRS appeared to be due to increases in water‐soluble choline compounds. This study illustrates the value of small localized voxels for differentiating regional chemical differences in tumors.

Enhanced elevation of corticosterone following arecoline administration to rats selectively bred for increased cholinergic function

Serum corticosterone levels were determined following administration of the cholinergic agonist arecoline (4 mg/kg) to rats selectively bred for differences in cholinergic function. The Flinders Sensitive Line (FSL) of rats exhibited both greater suppression of behavioural activity and enhanced elevation fo serum corticosterone than the Flinders Resistant Line of rats. These enhanced responses to arecoline in the FSL rats parallel those reported in depressed humans, suggesting that these rats may provide a new animal model of affective disorders.

Changes in presynaptic release of acetylcholine during development of tolerance to the anticholinesterase, DFP.

Abstract: The rat myenteric plexus was used as a peripheral model for studying muscarinic modulation of acetylcholine (ACh) release from presynaptic muscarinic neurons during development of tolerance to the anticholinesterase agent, diisopropylfluorophosphate (DFP). DFP in arachis oil was administered subcutaneously to intact animals according to both acute and chronic regimens, with arachis oil injections serving as controls. Postmortem analyses showed that the mean AChE activity level in whole brain was reduced under all DFP conditions to 18.0 ± 1.4% when compared with the control level. After 10 days of DFP treatment, the AChE level was 22.3 ± 2.1% of control in the myenteric plexus. There were no significant differences among the treatment groups in resting ACh release. Release evoked by electrical stimulation (difference between stimulated and resting release) in the absence of atropine, i.e., “basal rate,” for strips taken at various times after a single injection of DFP did not differ from that for strips from animals receiving arachis oil only. However, basal release for strips from chronically treated subjects was significantly greater than that of controls (p < 10−3), although not different from each other. Analysis of variance (ANOVA) for repeated measures showed that there existed a highly significant atropine dependency in strips from all treatments when they were stimulated in concentrations of atropine from 10−9 to 10−5M (p < 10−10). Further analyses established that the increases in rates of evoked ACh release as concentrations of atropine increased were similar for strips from chronically treated DFP and arachis oil animals. The overall results show that a significant effect of chronic depression in AChE activity is disinhibition of ACh‐evoked release from presynaptic neurons. This is consistent with the model in which muscarinic autoreceptors control evoked ACh release. The results are also consistent with time parameters for maximum decreases in [3H]quinuclidinyl benzilate binding in brain during chronic DFP treatments such as the regimens used in the present experiments.

Development of behavioral tolerance: A search for subcellular mechanisms

Development of behavioral tolerance is one of the processes by which living organisms adjust to changes in their internal and external environments. The search for neurochemical mechanisms underlying such processes requires the testing of many hypotheses. The present study was designed to examine the possible involvement of certain subcellular events. The concentrations of acetylcholine (ACh) and choline (Ch), the high-affinity transport of Ch, and the rate of synthesis of ACh were measured in synaptosomes prepared from the brains of rats. The assays were made at critical times during the acute changes in behavior induced by administration of the anticholinesterase, di-isopropylfluorophosphate, and during the development of behavioral tolerance to this compound as chronicity of administration continued. No statistically significant differences were found among treatment groups in the total concentration of ACh or Ch, the synthesis of ACh, or the high-affinity transport of Ch. These results, plus evidence from previous experiments, indicate that the development of behavioral tolerance does not relate to the factors studied. Consequently, alternative mechanisms should be considered. In addition to changes in cholinergic (muscarinic) receptors already shown to occur concomitantly with the development of behavioral tolerance, it is suggested that the possible involvement of mechanisms controlling release of ACh should be studied.

Behavioral and physiological effects associated with changes in muscarinic receptors following administration of an irreversible cholinergic agonist (BM 123).

Previous work in our laboratory has shown that the aziridinium ion of BM 123 (N-[4(2-chloroethylmethylamino)-2-butynyl]-2 pyrrolidone) is a potent and selective muscarinic agonist and binds irreversibly to muscarinic receptors (mAChR). The present series of experiments was designed to study the effects of BM 123 on behavioral and physiological variables known to be sensitive to manipulations of the cholinergic neurotransmitter system. BM 123 was injected into the tail vein of Sprague-Dawley rats, reducing mAChR to approximately 10% of normal as judged by [3H](−)QNB binding. Oxotremorine was injected IV for purposes of comparison. Behavioral and physiological variables were measured daily for 26 days. Physiological variables (e.g., tremor, chromodacryorrhea, salivation, and temperature) showed effects in less than 5 min after injection and returned to their pretreatment baselines within minutes. Nociceptive thresholds, dependent on sensory-perceptual processes, showed peak changes of approximately +230% and returned to normal within hours. Motoric responses, i.e., drinking and general activity, recovered in 3–4 days. Learned responses and those requiring temporal discrimination took 8–11 days to recover and were the only responses paralleling the return of the mAChRs to their normal levels. Changes elicited by oxotremorine recovered more rapidly than those elicited by BM 123. The results suggest that the different variables measured are dependent on different densities of functional receptors. Implications for a theoretical model are discussed.

Acetylcholine and choline dynamics provide early and late markers of traumatic brain injury

We assessed acetylcholine (ACh) and choline (Ch) dynamics 2.5 h, 1, 4 and 14 days after cerebral cortex impact injury or craniotomy only in adult male Sprague-Dawley rats. Cortical endogenous ACh (D0ACh), endogenous free Ch (D0Ch), deuterium-labeled Ch (D4Ch), and ACh synthesized from D4Ch (D4ACh) were measured by gas-chromatography mass-spectrometry after intravenous injection of D4Ch followed in 1 min by microwave fixation of the brain. D0Ch increased in and around the impact up to 700% of control within 1 day after trauma. Smaller D0Ch increases were found in the cortex contralateral to the impact and in both hemispheres after craniotomy only. D4Ch contents increased to 200% in the impact and surrounding regions 4-14 days post-trauma, with lower increases 2.5 h post-trauma. D0ACh decreased at all times post-trauma in the impact center, and initially in the periphery and adjacent regions with a recovery at 14 days. Similar D0ACh decreases, although of lesser extent and magnitude were present in the craniotomy only group. D4ACh showed a peak at one day post-trauma in all regions studied in the impact and craniotomy groups. In conclusion, D0Ch tissue level was an early marker of trauma, while 14 days after trauma Ch uptake from blood was enhanced in and around the traumatized cortex. Craniotomy by itself induced a generalized increase in ACh turnover 1 day after this minimal trauma. Choline acetyltransferase activity was reduced in the impact center region but not affected in the adjacent and contralateral regions or by craniotomy.

Global in vivo replacement of choline by N-aminodeanol. Testing a hypothesis about progressive degenerative dementia: I. Dynamics of choline replacement

Severe disruption of certain cholinergic pathways is a characteristic feature of Alzheimers disease. Attempts to establish animal models by interfering with cholinergic function have not been very successful. We now present data which show a substantial and progressive replacement of free and phospholipid-bound choline by the novel choline isostere N-amino-N,N-dimethylaminoethanol during its dietary administration in place of choline. Free choline in blood fell to approximately 20% of controls after 10 to 30 days on diet. Phospholipid-bound choline in plasma was reduced to less than 15%, and in erythrocytes to about 22%. After 120 days of diet free and bound choline were reduced in most tissues to approximately 30% of controls. Only liver retained more than 80% of free choline. Acetylcholine was decreased to 33 to 50% of control. Total true and false transmitter in experimental animals was in all tissues less that acetylcholine in controls, suggesting that muscarinic transmission would be impaired. Moderate reduction of choline acetyltransferase activity was seen in striatum and myenteric plexus, and of QNB-binding in hippocampus, striatum and myenteric plexus.