Ajit J. Shah

High-performance liquid chromatography/tandem mass spectrometric assay for the simultaneous measurement of dopamine, norepinephrine, 5-hydroxytryptamine and cocaine in biological samples

A rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed for the measurement of dopamine (DA), 5-hydroxytryptamine (5HT) and norepinephrine (NE) in brain microdialysates. The assay has also been utilised for the simultaneous measurement of these neurotransmitters and cocaine in brain dialysates. The neurotransmitters and cocaine were resolved in a single 4-min run using a binary gradient elution profile. The analytes were detected using tandem mass spectrometry in the positive ion electrospray mode. The limits of detection for DA, NE, 5HT and cocaine were 200, 1000, 900 pM and 1 pg ml(-1), respectively.

Selective antagonism at dopamine D3 receptors enhances monoaminergic and cholinergic neurotransmission in the rat anterior cingulate cortex

Recent neuroanatomical and functional investigations focusing on dopamine (DA) D3 receptors have suggested a potential role of this receptor in psychiatric diseases such as schizophrenia and drug dependence. In line with the key role of the prefrontal cortex in psychiatric disorders, the present study aimed at assessing the effects of the acute systemic administration of the selective DA D3 receptor antagonist SB-277011-A on the in vivo extracellular levels of monoamines (DA, norepinephrine (NE), and serotonin (5-HT)) and acetylcholine (ACh) in the anterior cingulate subregion of the medial prefrontal cortex. The in vivo neurochemical profile of SB-277011-A (10 mg/kg, i.p.) in the anterior cingulate cortex was compared with both typical and atypical antipsychotics including clozapine (10 mg/kg, s.c.), olanzapine (10 mg/kg, s.c.), sulpiride (10 mg/kg, s.c.), and haloperidol (0.5 mg/kg, s.c.). The acute administration of SB-277011-A, clozapine, and olanzapine produced a significant increase in extracellular levels of DA, NE, and ACh without affecting levels of 5-HT. Sulpiride also significantly increased extracellular DA, but with a delayed onset over SB-277011-A, clozapine, and olanzapine. In contrast, haloperidol failed to alter any of the three monoamines and ACh in the anterior cingulate cortex. These findings add to a growing body of evidence suggesting a differentiation between typical and atypical antipsychotic drugs (APDs) in the anterior cingulate cortex and a role of DA D3 receptors in desired antipsychotic drug profile. Similar to their effects on DA and NE, SB-277011-A, clozapine, and olanzapine increased extracellular levels of ACh, whereas haloperidol and sulpiride did not alter ACh. The results obtained in the present study provide evidence of the important role of DA D3 receptors in the effect of pharmacotherapeutic agents that are used for the treatment of psychiatric disorders such as schizophrenia and drug dependence.

Amino acid neurotransmitters: separation approaches and diagnostic value

Amino acids in the central nervous system can be divided into non-neurotransmitter or neurotransmitter depending on their function. The measurement of these small molecules in brain tissue and extracellular fluid has been used to develop effective treatment strategies for neuropsychiatric and neurodegenerative diseases and for the diagnosis of such pathologies. Here we describe the separation and detection techniques that have been used for the measurement of amino acids at trace levels in brain tissue and dialysates. An overview of the function of amino acid transmitters in the brain is given. In addition, the type of sampling techniques that are used for the determination of amino acid levels in the brain is described.

High-performance liquid chromatography/tandem mass spectrometry assay for the rapid high sensitivity measurement of basal acetylcholine from microdialysates

A high-throughput liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the analysis of acetylcholine (ACh) in brain dialysates. This separation of ACh is based on cation exchange chromatography with elution buffer consisting of a mixture of ammonium acetate, ammonium formate and acetonitrile. Using isocratic separation conditions, ACh was resolved within a minute and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for ACh was found to be 1 fmol on column with a S/N ratio of 3:1. The assay has been used routinely for the measurement of ACh in brain dialysates from awake freely moving rats. Furthermore, separation conditions were modified to allow simultaneous measurement of ACh and the acetylcholine esterase inhibitor, neostigmine.

Development and application of a sensitive high performance ion-exchange chromatography method for the simultaneous measurement of dopamine, 5-hydroxytryptamine and norepinephrine in microdialysates from the rat brain.

A high performance liquid chromatography (HPLC) method based on cation exchange separation has been developed for the measurement of dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) in microdialysates. The separation conditions have been optimised for using electrochemical detection. All three bioamines were resolved in less than 22 min using isocratic conditions. The optimum oxidation potential for the three bioamines was found to be +0.4 V vs. in situ Ag/AgCl reference electrode. Linear regression analysis of HPLC-peak area as a function of concentrations in the range 1-50 ng x ml(-1) gave coefficients of correlation between 0.998 and 0.999. The limit of detection for DA, 5-HT and NE was found to be between 50 and 100 pg x ml(-1) with a signal to noise ratio of 3:1. The method has been applied to the simultaneous measurement of the three monoamines in microdialysates from the medial prefrontal cortex under basal conditions and following the administration of the antipsychotic drug clozapine (10 mg x kg(-1) s.c.).

Fluoxetine administration modulates the cytoskeletal microtubular system in the rat hippocampus

A number of studies suggest that stressful conditions can induce structural alterations in the hippocampus and that antidepressant drugs may prevent such deficits. In particular, the selective serotonin reuptake inhibitor (SSRI) fluoxetine was more effective in modulating different neuronal plasticity phenomena and related molecules in rat hippocampus. Cytoskeletal microtubule dynamics are fundamental to dendrites and axons remodeling, leading to the hypothesis that fluoxetine may affect the microtubular system. However, despite reports of stress‐induced alterations in microtubule dynamics by different stressors, only few studies investigated the in vivo effects of antidepressants on microtubules in specific rat brain regions. The present study investigated the dose‐related (1, 5, or 10 mg/kg i.p.) effects of acute and chronic (21 days) treatments with fluoxetine on the ratio of hippocampal α‐tubulin isoforms which is thought to reflect microtubule dynamics. Western Blot analysis was used to quantify α‐tubulin isoforms, high‐performance liquid chromatography and fluorescence detection was used to measure ex vivo monoamine metabolism. The results showed that acute fluoxetine increased the stable forms acetylated and detyrosinated α‐tubulin. Conversely, chronic fluoxetine decreased acetylated α‐tubulin, indicative of increased microtubule dynamics. The neuron‐specific Δ2‐Tubulin was increased by chronic fluoxetine indicating neuronal involvement in the observed cytoskeletal changes. Although acute and chronic fluoxetine similarly altered serotonin metabolism by inhibition of serotonin reuptake, this showed no apparent correlation to the cytoskeletal perturbations. Our findings demonstrate that fluoxetine administration modulates microtubule dynamics in rat hippocampus. The cytoskeletal effect exerted by fluoxetine may eventually culminate in promoting events of structural neuronal remodeling. Synapse 63:359–364, 2009.

Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN-). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2-12.5 microM). The limit of detection for CBI derivatives of amino acids was in the range 5-20 fmol (S/N=2) using a 5 microl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.

Evaluation of expression and function of the H+/myo-inositol transporter HMIT

BackgroundThe phosphoinositide (PIns) signalling pathway regulates a series of neuronal processes, such as neurotransmitter release, that are thought to be altered in mood disorders. Furthermore, mood-stabilising drugs have been shown to inhibit key enzymes that regulate PIns production and alter neuronal growth cone morphology in an inositol-reversible manner. Here, we describe analyses of expression and function of the recently identified H+/myo-inositol transporter (HMIT) investigated as a potential regulator of PIns signalling.ResultsWe show that HMIT is primarily a neuronal transporter widely expressed in the rat and human brain, with particularly high levels in the hippocampus and cortex, as shown by immunohistochemistry. The transporter is localised at the Golgi apparatus in primary cultured neurones. No HMIT-mediated electrophysiological responses were detected in rat brain neurones or slices; in addition, inositol transport and homeostasis were unaffected in HMIT targeted null-mutant mice.ConclusionTogether, these data do not support a role for HMIT as a neuronal plasma membrane inositol transporter, as previously proposed. However, we observed that HMIT can transport inositol triphosphate, indicating unanticipated intracellular functions for this transporter that may be relevant to mood control.

Chronic fluoxetine differentially modulates the hippocampal microtubular and serotonergic system in grouped and isolation reared rats

Social isolation from weaning in rats produces behavioural and hippocampal structural changes at adulthood. Here, rats were group or isolation reared for eight-weeks. Following the initial four-week period of rearing, fluoxetine (10 mg/kg i.p.) was administered for 28 days. Changes in recognition memory, hippocampal monoamines, and cytoskeletal microtubules were investigated. Isolation-rearing for four- or eight-weeks produced recognition memory deficits that were not reversed by fluoxetine. Eight-weeks of isolation decreased alpha-tubulin acetylation (Acet-Tub) and the tyrosinated/detyrosinated alpha-tubulin ratio (Tyr/Glu-Tub), suggesting major alterations in microtubule dynamics and neuronal plasticity. In grouped rats, fluoxetine decreased Acet-Tub without changes in Tyr/Glu-Tub. In isolates, fluoxetine did not affect Acet-Tub but increased Tyr/Glu-Tub. Finally, fluoxetine altered serotonin metabolism in grouped, but not in isolated animals. Therefore, isolation-rearing changes the hippocampal responses of the serotonergic and microtubular system to fluoxetine. These findings show that early-life experience induces behavioural changes paralleled by alterations in cytoskeletal and neurochemical functions.

High-performance liquid chromatography/tandem mass spectrometry assay for the determination of 1-methyl-4-phenyl pyridinium (MPP+) in brain tissue homogenates

A high-throughput liquid chromatography/tandem mass spectrometry method has been developed for the quantitative assessment of 1-methyl-4-phenylpyridinium (MPP+) in brain tissue samples. This separation is based on reversed phase chromatography using formic acid and acetonitrile as the mobile phase. Using gradient separation conditions, MPP+ was resolved within 5 min and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for MPP+ was found to be 1 fmol on column with a signal to noise ratio of 3:1. The assay has been used routinely in our laboratory for the measurement of MPP+ levels in brain tissue from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and can be used to distinguish neuroprotective efficacy and monoamine oxidase inhibition.