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Objective: Separation of tricyclic compounds sets the keystone for determining parent drug to metabolite concentration ratios and analysing impurities. The combined effects of acetonitrile composition and pH of the mobile phase on the separation of amitriptyline and nortriptyline by reversed-phase high-performance liquid chromatography (RP-HPLC) are presented. Methods: A series of RP-HPLC triplicate runs were carried out using acetonitrile and a phosphate buffer as the mobile phase and a Kinetex® C18 LC Column as the stationary phase using an Agilent 1260 Infinity Series® II liquid chromatography system with UV/visible detection. The stationary phase, column temperature, injection volume and flow rate were kept unchanged during analysis. Mobile phase composition and pH were varied to observe impact on peak shape, resolution and retention time, taking into consideration green analytical chemistry aspects. Results: Optimal chromatographic outcomes were achieved when using the mobile phase made up of 35% acetonitrile and 65% buffer at a pH of 5.6. These conditions resulted in nortriptyline and amitriptyline eluting at 4.66 min and 5.92 min respectively. Increasing the organic modifier content of the mobile phase to 40% completed separation within a run time of 4 min with comparable resolution. The 2 min gained by increasing 5% acetonitrile may not be justified due to potential implications on greening laboratory practices. Conclusion: Reversed-phase chromatography embodies a simple method for the separation of compounds that are similar in structure. Attuning the percentage of organic modifier and buffer pH provides acceptable retention times, without compromising resolution between neighbouring peaks., peer-reviewed

In Alzheimer's disease the loss of cholinergic neurons is often thought to be essential but cholinesterase inhibitors have been helpful only for a part of patients. Experimental and clinical evidence supports the importance of noradrenergic neurons in arousal but also in attention and memory. Some of these neurones seem to end in contact with cholinergic neurons. In animal studies, both a2-adrenoceptor agonists and antagonists have been reported to improve some cognitive functions. The reported clinical tests with clonidine have been negative like our experiments with a less sedative a2-agonist, guanfacine, in rats. An injection of atipamezole (ATI), a2-adrenoceptor antagonist (Farmos Group) decreased total motor activity, rearing and grooming in rats in the open-field test. In the Morris water maze, after high doses (>3mg/ kg) of ATI the rats did not swim, after Iow doses they made less errors. In the appetitive spatial memory test, after low doses of ATI, the rats showed slightly less forgetting than the controls. In the passive avoidance test, acute treatment with ATI improved the performance of rats. ATI enhanced the EEG of rats decreasing slow wave spikes like cholinergic drugs do. Autoradiographic localization of ATI binding sites in rat brain slices indicated that those sites are distributed throughout the whole brain and have a similar location to other imidazole containing a2-adrenoceptor antagonists. Several neurotransmitters may be involved in the actions of ATI since it seemed to release noradrenaline and serotonin in brain., N/A

Neurons in the rat frontal cortex demonstrate altered firing rates in response to conditioned stimuli. Experiments were designed to test the hypothesis that the basal forebrain cholinergic systera is involved in the generation of conditioned neuronal responses in the rat frontal cortex. Most of the cholinergic innervation to the frontal cortex is supplied by projections frora the nucleus basalis raagnocellularis (nBM), A two-second tone cue imraediately followed by rewarding medial forebrain bundle stimulation was used to elicit the conditioned neuronal responses in the following experiraents. Experiment One was performed to determine the effects of inhibiting the firing of neurons in the nBM region on the conditioned responses. This was presuraably accomplished by microinjecting procaine, a local anesthetic, or GABA, an inhibitory neurotransraitter, into the nBM region. Procaine antagonized the conditioned responses of 86% of the cortical single units tested and GABA antagonized the responses of 81% of the units. Experiment Two was designed to investigate the effects of blocking cholinergic receptors on the conditioned responses of frontal cortex neurons. Microinjection of atropine, a cholinergic antagonist, into the frontal cortex suppressed the conditioned responses of 21 of 24 cortical single units. The nBM region was lesioned with kainic acid, a neurotoxin, in Experiment Three. The lesion resulted in a significant decrease in choline acetyltransferase activity in the frontal cortex ipsilateral to the nBM lesion. Only 25% of the neurons recorded in the frontal cortex on the side of the nBM lesion exhibited conditioned responses. This was significantly lower than the percentage of neurons that exhibited conditioned responses (70%) in the cortex of untreated animals. The firing rates of units in the nBM region were monitored during the cue-event paradigm in Experiment Four. Of the 38 unit recordings from the nBM region, 28 (74%) exhibited conditioned responses. The results from the four experiments together provide strong evidence for a role of the basal forebrain cholinergic system in the generation of conditioned single unit responses in the frontal cortex.

The enzyme choline 0-acetyltransferase (EC 2.3.1.6, ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine in central and peripheral cholinergic nerve tissue. Traditionally, ChAT was thought to exist exclusively as a soluble, cytoplasmic enzyme in nerve endings. However, several experimental results suggest that a form of ChAT exists which is more closely associated with the membranes of nerve endings. For example, although acetylcholine is stored in both the cytoplasm and the vesicles of nerve endings, these two pools are metabolized independently and supply acetylcholine for different modes of release. Cytoplasmic acetylcholine has never been demonstrated to be transported into synaptic vesicles, but choline formed from hydrolyzed acetylcholine may be transported into vesicles for synthesis of vesicular acetylcholine. The objective of this study was to determine whether membrane-bound forms of ChAT exist, of which one form may be more closely associated with the vesicles. Three fraction of ChAT were solubilized from a crude vesicular membrane fraction of brain, by washing the membranes with three solutions of increasing chaotrophic nature: 100 mM sodium phosphate buffer, pH 7.4 (NaP), 500 mM NaCl, and 2% Tritón DN-65. A variety of biochemical, anatomical, biophysical, developmental, and pharmacological properties of the three fractions were then examined to determine whether they differ from each other. NaP soluble ChAT, which is traditionally thought to be a soluble (cytoplasmic) enzyme, differed from the other two fractions of ChAT with respect to pH profile, heat inactivation, cardiolipin inhibition, hydrophobic chromatography on phenyl-Sepharose, and developmental pattern. NaCl soluble ChAT differed from the other two fractions with respect to K for choline, pH profile, anatomical distribution, sensitivity to septal-hippocampal lesion, heat inactivation, hydrophobic chromatography on phenyl-Sepharose, developmental pattern, and the effect of atropine administration. Tritón soluble ChAT differed from the other two fractions with respect to ability to acetylate homocholine, heat inactivation, and developmental pattern. The NaP soluble forra of ChAT may supply the cytoplasmic pool of acetylcholine, while it appears that the Tritón soluble form of ChAT may synthesize vesicular acetylcholine. The physiological role of NaCl soluble ChAT is more uncertain, but the results support the possibility that this form may be a precursor for one or both of the other two forms of ChAT.