Glucose is an adequate energy substrate for the depolarizing action of GABA and glycine in the neonatal rat spinal cord in vitro.

In vitro studies have repeatedly demonstrated that the neurotransmitters γ-aminobutyric acid (GABA) and glycine depolarize immature neurons in many areas of the CNS, including the spinal cord. This widely accepted phenomenon was recently challenged by experiments showing that the depolarizing action of GABA on neonatal hippocampus and neocortex in vitro was prevented by adding energy substrates (ES), such as the ketone body metabolite dl-ß-hydroxybutyric acid (DL-BHB), lactate, or pyruvate to the artificial cerebrospinal fluid (ACSF). It was suggested that GABA-induced depolarizations in vitro might be an artifact due to inadequate energy supply when glucose is the sole energy source, consistent with the energy metabolism of neonatal rat brain being largely dependent on ESs other than glucose. Here we examined the effects of these ESs (DL-BHB, lactate, pyruvate) on inhibitory postsynaptic potentials (IPSPs) recorded from neonatal rat lumbar spinal cord motoneurons (MNs), in vitro. We report that supplementing the ACSF with physiologic concentrations of DL-BHB, lactate, or pyruvate does not alter the reversal potential of IPSPs (E(IPSP)). Only high concentrations of pyruvate hyperpolarized E(IPSP). In addition, the depolarizing action of GABA on primary afferent terminals was not affected by supplementing the ACSF with ES at physiologic concentrations. We conclude that depolarizing IPSPs in immature MNs and the primary afferent depolarizations are not caused by inadequate energy supply. Glucose at its standard concentration appears to be an adequate ES for the neonatal spinal cord in vitro.

Effects of topography on the functional development of human neural progenitor cells.

We have fabricated a topographical substrate with a packed polystyrene bead array for the development of cell-based assay systems targeting voltage-gated calcium channels (VGCCs). Human neural progenitor cells (H945RB.3) cultured on both flat and topographical substrates were analyzed in terms of morphological spreading, neuronal commitment, resting membrane potential (V(m)) establishment and VGCC function development. We found, by SEM imaging, that arrayed substrates, formed with both sub-micrometer (of 0.51 microm in mean diameter) and micrometer (of 1.98 microm in mean diameter) beads, were capable of promoting the spreading of the progenitor cells as compared with the flat polystyrene surfaces. With the micrometer beads, it was found that arrayed substrates facilitated the neural progenitor cells' maintenance of less negative V(m) values upon differentiation with bFGF starvation, which favored predominant neuronal commitment. Almost all the progenitor cells were responsive to 50 mM K(+) depolarization with an increase in [Ca(2+)](i) either before or upon differentiation, suggesting the expression of functional VGCCs. Compared to the flat polystyrene surfaces, microbead arrayed substrates facilitated the development of higher VGCC responsiveness by the progenitor cells upon differentiation. The enhancement of both VGCC responsiveness and cell spreading by arrays of micrometer beads was most significant on day 14 into differentiation, which was the latest time point of measurement in this study. This study thus rationalized the possibility for future substrate topography engineering to manipulate ion channel function and to meet the challenge of low VGCC responsiveness found in early drug discovery.

Mode of action of piperovatine, an insecticidal piperamide isolated from Piper piscatorum (Piperaceae), against voltage-gated sodium channels.

Piperamides, which are secondary metabolites in the genus Piper, have potent insecticidal activity, and have thus inspired the development of novel insecticides. In this study, piperovatine, a piperamide from Piper piscatorum (Piperaceae), was investigated using a two-electrode voltage clamp to clarify its detailed mode of action against voltage-gated sodium channels, a classic target. In Xenopus oocytes expressing voltage-gated sodium channels from German cockroach (Blattella germanica), piperovatine induced inward currents depending on repetitive openings. For instance, maximal currents were generated with 10 µM piperovatine following 100 trains of depolarizing pulses with frequency 25 Hz. Piperovatine also shifted the half-activation voltage after conditioning pulses from -35 mV to -45 mV. In addition, piperovatine-modified currents were correlated with not only the number of prior conditioning pulses but also the proportion of activated channels. Finally, piperovatine was found to stabilize voltage-gated sodium channels in the fast-inactivated state after opening, and inhibit transition to the slow-inactivated state. These results suggest that piperovatine preferably binds to activated channels and stabilizes voltage sensors at the conformation acquired during depolarization.

Qualitative identification of doxacurium and its breakdown products in postmortem fluids by liquid chromatography-tandem mass spectrometry.

During a death investigation at the Office of the Cuyahoga County Coroner in Cleveland, OH, doxacurium became a drug of interest. The Coroner's Office enlisted the aid of the Federal Bureau of Investigation Laboratory for the doxacurium analysis. Following the request, a method for the extraction and qualitative analysis of the drug in biological fluids was developed. The procedure relies on a simple solid-phase extraction procedure followed by qualitative analysis with liquid chromatography-tandem mass spectrometry. During the development of the new analytical procedure, two breakdown products of doxacurium were detected. Structures for these breakdown products are proposed. This procedure was used to analyze heart blood, cerebrospinal fluid, and bile specimens from the decedent. Doxacurium and its breakdown products were identified in all three specimens.

Anesthesiologist suicide with atracurium.

Atracurium is a nondepolarizing skeletal muscle relaxant used to facilitate endotracheal intubation and to induce skeletal muscle relaxation during surgery or mechanical ventilation. The drug undergoes a spontaneous non-enzymatic biotransformation, yielding laudanosine and an acrylate moiety. This report documents the case of a 45-year-old anesthesiologist who was found dead at the hospital where he worked. The victim was known to be depressed and undergoing treatment with venlafaxine. An empty syringe was found near the body. Toxicological analysis revealed the presence of laudanosine in the syringe, 0.6 mg/L of laudanosine in heart blood, 0.3 mg/L in urine, and 0.02 mg/L in vitreous humor. Meanwhile, concentrations of venlafaxine and O-desmethyl-venlafaxine, its active metabolite, were 0.7 and 1.1 mg/L in heart blood, 1.7 and 5.2 mg/L in urine, 0.5 and 0.7 mg/L in vitreous humor, and 400 and 20 mg in gastric content, respectively. All drugs and metabolites involved in the case were detected using gas chromatography with nitrogen-phosphorus detection (GC-NPD) and confirmed using GC-mass spectrometry in full scan mode after solid-phase extraction using Bond-Elut Certify columns. Additional high-performance liquid chromatography coupled to diode-array detection screening also obtained the same results. Quantitation of laudanosine and venlafaxine together with its metabolite was carried out using GC-NPD. No other drugs, including ethanol, were detected. Recoveries for laudanosine and venlafaxine were 89% and 86%, respectively, at 0.5 mg/L; intraday and interday precisions were 2% and 6%, and 3% and 7%, respectively; and limits of detection and quantitation were 6 and 20 ng/mL and 18 and 59 ng/mL, respectively. The linearity of the blood calibration curves was excellent for both drugs with r(2) values of > 0.999 (range 0.1-2.0 mg/L). Based on the autopsy findings, case history, and toxicology results, the forensic pathologists ruled that the cause of death was an overdose of atracurium, and the manner of death was suicide.

How important is the butyrylcholinesterase level for cesarean section?

OBJECTIVES: Butyrylcholinesterase (BChE), commonly known as pseudocholinesterase or non-neural cholinesterase, hydrolyzes neuromuscular blocker agents containing choline esters such as succinylcholine that is widely used in rapid sequence induction (RSI) for general anesthesia. The aim of this study is to compare plasma BChE levels and investigate the affects and relationship of succinylcholine on BChE levels in preeclamptic, gestational diabetic and healthy pregnants. STUDY DESIGN: We designed a prospective, controlled, pilot single-center study. Thirty (n=30) pregnant women who were scheduled for cesarean section under general anesthesia (refusal of regional anesthesia) with RSI involved. Group 1 included ten (n=10) preeclamptic pregnancies, Group 2 included ten (n=10) gestational diabetic (GD) pregnancies and Group 3 included ten (n=10) healthy pregnancies. MAIN OUTCOME MEASURES: BChE levels of all patients were measured prior to the initiation of cesarean section. Train-of-four recovery of 90% (TOF T1) was used to monitor the degree of neuromuscular block beginning from the administration of succinylcholine. RESULTS: No statistically significant difference was found between the groups comparing BChE levels and the duration between tracheal intubation and formation of TOF T1 (p>0.05). CONCLUSIONS: As similar results were gathered from normal and high-risk pregnancies (preeclamptic pregnancy or gestational diabetic pregnancy) who underwent cesarean section under general anesthesia, we believe that succinylcholine is still neuromuscular agent of choice in cesarean section.

Purification of active synaptic vesicles from the electric organ of Torpedo californica and comparison to reserve vesicles.

At least two distinguishable forms of synaptic vesicles exist, the active and reserve, but the reserve form is studied most because it has been difficult to purify the active vesicles. In the work reported here the active vesicles (termed VP2) were highly enriched from the electric organ of Torpedo californica by an improved method developed for the reserve vesicles (termed VP1) with the addition of density gradient centrifugation based on Percoll. No significant differences between the vesicular types were found in the amounts of SV1, SV2, and SV4 epitopes and P-type and V-type ATPase activities. The buoyant densities (g/ml) of VP1 and VP2 vesicles were determined by centrifugation in isosmotic sucrose (1.051, 1.069), Percoll (1.034, 1.040), and glycerol (1.087, 1.090) gradients. The radii were determined by dynamic quasi-elastic laser light-scattering to be (56.6 +/- 10.8) nm and (55.0 +/- 12.7) nm. For both vesicular types the volume of excluded sucrose is only about 37% of the volume of excluded Percoll, indicating that the surfaces are rough. Approx. 51% of the VP1 and 32% of the VP2 vesicular volumes are 'osmotically active' water that is exchangeable with glycerol. The different buoyant densities and amounts of osmotically active water in VP1 and VP2 vesicles probably are due to the different internal solutes. Previously observed differences in acetylcholine active transport and vesamicol binding by VP1 and VP2 synaptic vesicles cannot be explained by major alterations in the protein composition or conformation of the membranes in the two types of vesicles.

New transport assay demonstrates vesicular acetylcholine transporter has many alternative substrates.

The acetylcholine-binding site in vesicular acetylcholine transporter faces predominantly toward the outside of the vesicle when resting but predominantly toward the inside when transporting. Transport-related reorientation is detected by an ATP-induced decrease in the ability of saturating substrate to displace allosterically bound [(3)H]vesamicol. The assay was used here to determine whether structurally diverse compounds are transported by rat VAChT expressed in PC12(A123.7) cells. Competition by ethidium, tetraphenylphosphonium and other monovalent organic cations with [(3)H]vesamicol is decreased when ATP is added, and the effect depends on proton-motive force. The results indicate that many organic molecules carrying +1 charge are transported, even though the compounds do not resemble acetylcholine in structural details.

Widespread depolarization during expiration: a source of respiratory drive?

Respiration influences various pacemakers and rhythms of the body during inspiration and expiration but the underlying mechanisms are relatively unknown. Understanding this phenomenon is important, as breathing disorders, breath holding, and hyperventilation can lead to significant medical conditions. We discuss the physiological modulation of heart rhythm, blood pressure, sympathetic nerve activity, EEG, and other changes observed during inspiration and expiration. We also correlate the intracellular mitochondrial respiratory metabolic processes with real-time breathing and correlate membrane potential changes with inspiration and expiration. We propose that widespread minor hyperpolarization occurs during inspiration and widespread minor depolarization occurs during expiration. This depolarization is likely a source of respiratory drive. Further knowledge of intracellular and extracellular ionic changes associated with respiration will enhance ourunderstanding of respiration and its role as a modulator of cellular membrane potential. This could expand treatment options for a wide range of health conditions, such as breathing disorders, stress-related disorders, and further our understanding of the Hering-Breuer reflex and respiratory sinus arrhythmia.

Vesamicol receptor mapping of brain cholinergic neurons with radioiodine-labeled positional isomers of benzovesamicol.

UNLABELLED: Alzheimer's disease is characterized by progressive cerebral cholinergic neuronal degeneration. Radiotracer analogs of benzovesamicol, which bind with high affinity to the vesamicol receptor located on the uptake transporter of acetylcholine storage vesicles, may provide an in vivo marker of cholinergic neuronal integrity. Five positional isomers of racemic iodobenzovesamicol (4'-, 5-, 6-, 7-, and 8-IBVM) were synthesized, exchange-labeled with iodine-125, and evaluated as possible in vivo markers for central cholinergic neurons. Only two isomers, 5-IBVM (5) and 6-IBVM (10), gave distribution patterns in mouse brain consistent with cholinergic innervation: striatum >> hippocampus > or = cortex > hypothalamus >> cerebellum. The 24-h tissue-to-cerebellum concentration ratios for 5-IBVM (5) were 3-4-fold higher for striatum, cortex, and hippocampus than the respective ratios for 6-IBVM (10). Neither 8-IBVM (16) nor 4'-IBVM (17) exhibited selective retention in any of the brain regions examined. In the heart, only 5-IBVM (5) exhibited an atria-to-ventricles concentration ratio consistent with high peripheral cholinergic neuronal selectivity. The 7-IBVM (14) isomer exhibited an anomalous brain distribution pattern, marked by high and prolonged retention in the five brain regions, most notably the cerebellum. This isomer was screened for binding in a series of 26 different biological assays; 7-IBVM (14) exhibited affinity only for the delta-receptor with an IC50 of approximately 30 nM. Drug-blocking studies suggested that brain retention of 7-IBVM (14) reflects high-affinity binding to both vesamicol and delta-receptors. Competitive binding studies using rat cortical homogenates gave IC50 values for binding to the vesamicol receptor of 2.5 nM for 5-IBVM (5), 4.8 nM for 6-IBVM (10), and 3.5 nM for 7-IBVM (14). Ex vivo autoradiography of rat brain after injection of (-)-5-[125I]IBVM ((-)-[125I]5) clearly delineated small cholinergic-rich areas such as basolateral amygdala, interpeduncular nucleus, and facial nuclei. Except for cortex, regional brain levels of (-)-5-[123I]IBVM ((-)-[123I]5) at 4 h exhibited a linear correlation (r2 = 0.99) with endogenous levels of choline acetyltransferase. CONCLUSION: Vesamicol receptor mapping of cholinergic nerve terminals in murine brain can be achieved with 5-IBVM (5) and less robustly with 6-IBVM (10), whereas the brain localization of 7-IBVM (14) reflects high-affinity binding to both vesamicol and delta-receptors.

N-hydroxyalkyl derivatives of 3 beta-phenyltropane and 1-methylspiro[1H-indoline-3,4'-piperidine]: vesamicol analogues with affinity for monoamine transporters.

As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter ligand 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives of 3 beta-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4'-piperidine], 7, were synthesized and tested for binding in vitro. Although a few compounds displayed moderately high affinity for the vesicular acetylcholine transporter, no compound was more potent than the prototypical vesicular acetylcholine transporter ligand vesamicol. However, a few derivatives of 6 displayed higher affinity for the dopamine transporter than cocaine. We conclude that modification of the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter ligands.

Choline acetyltransferase activity and vesamicol binding in Rett syndrome and in rats with nucleus basalis lesions.

The decline in choline acetyltransferase activity has been identified previously within the brains of patients with Rett syndrome and Alzheimer's disease. The level of [3H]vesamicol binding to a terminal vesicular acetylcholine transporter is inversely related to the decline in cortical choline acetyltransferase activity in Alzheimer's disease, which may be due to compensatory processes within surviving cholinergic terminals. In order to investigate whether similar cholinergic compensatory processes are present in the Rett syndrome brain and are altered by normal aging, we investigated the density of cholinergic vesicular transporters in (i) the brains of Rett syndrome patients, and (ii) young and old rats with experimentally-induced cholinergic cell loss. In Rett syndrome, a significant decline in choline acetyltransferase activity within the putamen and thalamus was directly correlated with a decline in [3H]vesamicol binding. In both young and old rats, basal forebrain lesions decreased cortical choline acetyltransferase activity significantly, while [3H]vesamicol binding was unchanged. In contrast to young and old lesioned rats and patients with Alzheimer's disease, cholinergic cells in the brains of patients with Rett syndrome do not compensate for the loss of cholinergic cells by increasing acetylcholine vesicular storage.

Choline acetyltransferase activity and [3H]vesamicol binding in the temporal cortex of patients with Alzheimer's disease, Parkinson's disease, and rats with basal forebrain lesions.

[3H]Vesamicol binding was characterized in human brain post mortem. The number of binding sites was then determined in parallel with choline acetyltransferase activity in the temporal cortex of patients with Alzheimer's disease, demented and non-demented patients with Parkinson's disease, and in the cerebral cortex of rats with quisqualic acid lesions of the nucleus basalis magnocellularis. Whereas choline acetyltransferase activity decreased in patients with Alzheimer's or Parkinson's disease indicating loss of cholinergic innervation, the number of binding sites for [3H]vesamicol was the same as or higher than in controls. Similar results were obtained with the lesioned rats. It is suggested that the increase in binding sites may reflect compensatory regulation of the spared neurons at the level of the synaptic vesicle.

Cholinergic regulation of hippocampal brain-derived neurotrophic factor mRNA expression: evidence from lesion and chronic cholinergic drug treatment studies.

Quantitative in situ hybridization and northern blot analysis techniques were used to determine the effects of removal of the cholinergic input on levels and topographical distribution of brain-derived neurotrophic factor mRNA in the hippocampus of adult rats. First, the effects of partial and full fimbrial transections, which result in partial and near-total cholinergic deafferentation respectively, were compared. Twenty-one days after partial unilateral fimbrial transections, there were significant decreases in brain-derived neurotrophic factor mRNA expression throughout the hippocampal formation. Decreased expression of brain-derived neurotrophic factor mRNA was evident in all areas of localization within the hippocampal formation. The decreases amounted to 22-36% reductions compared with unlesioned control animals. Brain-derived neurotrophic factor mRNA levels were decreased to a greater extent (50-69%) following full unilateral fimbrial transections. Quantitative northern blot analysis indicated that hippocampal BDNF mRNA was decreased by 29 and 68%, three weeks after partial or full unilateral fimbrial transections, respectively. The extent of the reductions in brain-derived neurotrophic factor mRNA levels correlated with reductions in acetylcholinesterase staining density and cholinergic terminal density determined by quantitative autoradiographic analysis of [3H]vesamicol binding sites. Second, we found that chronic treatment with atropine (20 mg/kg per day for 14 days) decreased (by 54%) brain-derived neurotrophic factor mRNA levels in all areas of localization within the hippocampus. In contrast, chronic treatment with nicotine (1.18 mg/kg per day for 14 days), a treatment known to desensitize nicotinic receptors, did not affect brain-derived neurotrophic factor mRNA expression in the hippocampal formation. The findings provide evidence for cholinergic muscarinic regulation of brain-derived neurotrophic factor mRNA expression in the adult rat hippocampal formation and they suggest the existence of a tonic stimulation of brain-derived neurotrophic factor synthesis by the cholinergic afferents.

Intravenous anaesthetic agents. Pharmacokinetic-pharmacodynamic relationships.

The number and variety of intravenous anaesthetics available has increased dramatically in recent years. In order to administer these agents safely and rationally in the operating theatre, the anaesthesiologist needs to have a detailed understanding of their dose-response behaviour. The pharmacokinetic and pharmacodynamic profiles of these anaesthetics underlie this behaviour. In understanding and applying this information, an integrated approach involving combined pharmacokinetic and pharmacodynamic modelling has proved tremendously helpful. This approach has provided significant insight into the pharmacology of these drugs under conditions of normal and abnormal physiology. Intravenous anaesthetics may be classified as hypnotics, narcotics, or muscle relaxants. Through quantitative measures or drug effect, as provided by the electroencephalogram (EEG) in the former two classes or electromyography in the latter class, precise measures of end-organ (brain or neuromuscular junction) sensitivity have been generated using integrated modelling techniques. These values not only allow for dose potency comparisons within an anaesthetic class but, furthermore, may explain changes in drug response with ageing and disease. Incorporation within these models of an effect compartment, characterised by a rate constant for equilibration with plasma (keo), has proved to be essential in accurately describing the temporal lag between drug administration and effect. Quantitating the size of this lag has direct implications for the design of anaesthetic bolus and infusion regimens. Combined pharmacokinetic and pharmacodynamic modelling of intravenous anaesthetics provides a precise, rational basis for the clinical use of these classes of drugs.

Vesamicol analogues as sigma ligands. Molecular determinants of selectivity at the vesamicol receptor.

The present study compares the affinities of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1) and selected analogues of the latter at the vesamicol receptor (VR) with the corresponding affinities at sigma 1 and sigma 2 binding sites. For this study, the parent structure 1 was divided into three fragments: A (cyclohexyl), B (piperidyl) and C (phenyl). Vesamicol analogues were then selected to reflect structural modifications in these fragments. Consistent with earlier reports, vesamicol was found to exhibit nanomolar affinities at the VR and sigma 1 and sigma 2 sites, resulting in poor selectivity for the VR over the sigma sites. Vesamicol analogues characterized by an acyclic A-fragment showed moderate to low affinities at the VR and moderate to high affinities at sigma 1 and sigma 2 sites. As a result, many of these analogues showed poor selectivity for the VR. Replacement of the C4 carbon of 1 with a halobenzyl amine resulted in higher affinities at the VR coupled with moderate to low affinities at sigma 1 and sigma 2 sites. The introduction of a benzofused substituent at the C4 and C5 positions of 1 (compound 2) resulted in a 200-fold increase in affinity at the VR accompanied by a 5- to 6-fold decrease in affinity at sigma 1 and sigma 2 sites relative to the parent structure. Consequently, compound 2 showed 12,000-fold higher affinity at the VR than at sigma sites. Restricting the rotation of fragment C relative to B (by means of alkyl and alkenyl bridges) generally yielded analogues with subnanomolar affinities at the VR. The corresponding affinities of these spirofused conformationally restricted analogues were moderate to poor at sigma 1 and sigma 2 sites when fragment A was preserved. In contrast, the affinities at sigma 1 and sigma 2 sites were decreased 3- to 11-fold when fragment A was modified at position C4 and decreased up to 100-fold with benzofusion at the C4 and C5 positions of fragment A. Consequently, the spirofused analogues 15-19 were among the most selective VR ligands examined. Thus, the effect of conformational restriction in fragments A and B-C is to increase affinity at the VR while decreasing affinity at sigma 1 and sigma 2 sites, and thereby increasing selectivity for the VR over the sigma sites.

A critical histidine in the vesicular acetylcholine transporter.

The role of proton binding sites in the vesicular acetylcholine transporter was investigated by characterization of the pH dependence for the binding of [3H]vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol] to Torpedo synaptic vesicles. A single proton binds to a site with pKa 7.1 +/- 0.1, which is characteristic of histidine, to competitively inhibit vesamicol binding. The histidine-selective reagent diethylpyrocarbonate causes time-dependent inhibition of [3H]vesamicol binding with a rate constant only about 20-fold lower than for reaction with free histidine. Because its pH titration has a simple, ideal shape, this residue probably controls all pH effects in the transporter between pH 6-8. Inhibition of [3H]vesamicol binding by diethylpyrocarbonate was slowed by vesamicol but not acetylcholine, which binds to a separate site. The data suggest that a critical histidine with a pKa of 7.1 is unhindered when reacting with diethylpyrocarbonate. A conformational model for the histidine is proposed to explain why acetylcholine competes with protons but not with diethylpyrocarbonate. A conserved histidine in transmembrane helix VIII possibly is the histidine detected here.

Effect of N,N'-dicyclohexylcarbodiimide on the binding of vesamicol, an inhibitor of acetylcholine transport into synaptic vesicles.

Vesamicol is a highly potent inhibitor of active acetylcholine transport into isolated cholinergic vesicles from Torpedo. On the basis of transport kinetics and vesamicol sensitivity, we have shown that the acetylcholine transporter could be in an activated state even in the absence of a stimulated ATPase. In this preparation, N,N'-dicyclohexylcarbodiimide (DCCD), an hydrophobic carbodiimide, inactivates both ACh transport and vesamicol binding. Inhibition of vesamicol binding by DCCD is time dependent, saturable and prevented by vesamicol. DCCD first affected the affinity constant for vesamicol. Ki-value for DCCD lies in the micromolar range. These results imply that there is a DCCD reactive site within the ACh transporter and that it is located in an hydrophobic environment near the vesamicol binding site. SDS-gel electrophoresis after labelling of the vesicle membrane proteins with [14C]DCCD shows that radioactivity is mainly incorporated in a 15 kDa subunit. Time-course and concentration dependence of [14C]DCCD labelling and vesamicol inhibition do not coincide. Hence, the two processes are probably unrelated and the result rather points to another inactivation mechanism which can be an intramolecular cross link.

Interactions between the effects of basal forebrain lesions and chronic treatment with MDL 26,479 on learning and markers of cholinergic transmission.

The effects of ibotenic acid-induced basal forebrain lesions and treatment with the triazole MDL 26,479 on the acquisition of an operant visual conditional discrimination task and on [3H]hemicholinium-3 and [3H]vesamicol binding were examined. Lesioned animals required more training sessions to acquire the stimulus-response rules of this task. They also showed longer response latencies throughout the experiment. The effects of the treatment with MDL 26,479 (5 mg/kg; i.p. 60 min before each training session) interacted with the effects of the lesion, producing a decrease in the number of sessions required to perform above chance-level in lesioned but not in control animals. MDL 26,479 did not seem to produce immediate performance effects but interacted with the learning process. The lesions destroyed the cell bodies in the area of the substantia innominata, basal nucleus of Meynert, and the globus pallidus. The number of frontocortical cholinergic terminals as primarily indicated by hemicholinium-3 binding was reduced in lesioned animals; however, another measure of cholinergic terminals, vesamicol binding, was unchanged. Behavioral performance of animals correlated significantly with hemicholinium binding in the frontal cortex of the right hemisphere. The fact that the lesion delayed but did not block the acquisition of the task may have been a result of compensatory mechanisms in remaining cholinergic terminals as indicated by stable vesamicol binding. These data allow assumptions about the conditions for the demonstration of beneficial behavioral effects of MDL 26,479. They also suggest that the long-term effects of basal forebrain lesions on cortical cholinergic transmission remain unsettled.

Pharmacological characterization of the vesamicol analogue (+)-[(125)I]MIBT in primate brain.

The vesamicol analogue, meta-[(125)I]iodobenzyltrozamicol [(+)-[(125)I]MIBT] was evaluated as a probe for the in vitro labeling of the vesicular acetylcholine transporter in primate brain. In the striatum, (+)-[(125)I]MIBT bound a single high-affinity site with a Kd value of 4.4 +/- 0.7 nM. Competition for (+)-[(125)I]MIBT binding to the striatum by a group of vesamicol analogues displayed a pharmacological profile similar to the rank order of potency previously observed for the vesicular acetylcholine transporter on Torpedo synaptic vesicles. High-affinity binding of (+)-[(125)I]MIBT in the occipital cortex was characterized by a Kd value of 4.6 +/- 1.1 nM. However, the rank order of potency for inhibition of (+)-[(125)I]MIBT binding to the occipital cortex by the same test compounds differed from that observed in the striatum. The results suggest that (+)-[(125)I]MIBT is a reliable probe of the vesicular acetylcholine transporter in primate striatum, but its binding in primate occipital cortex is more complex.