Takashi Yoshitake

Mephedrone, compared with MDMA (ecstasy) and amphetamine, rapidly increases both dopamine and 5-HT levels in nucleus accumbens of awake rats

BACKGROUND AND PURPOSE The designer drug 1‐(4‐methylphenyl)‐2‐methylaminopropan‐1‐one (4‐methylmethcathinone, mephedrone) is reported to possess psychostimulant, entactogenic and hallucinogenic effects. The purpose of this study was to examine the effects of acute administration of mephedrone on extracellular levels of dopamine (DA) and 5‐HT in the nucleus accumbens of awake rats and compare these effects with those induced by 3,4‐methylenedioxymethamphetamine (MDMA, ecstasy) and amphetamine.

Galanin Is a Potent In Vivo Modulator of Mesencephalic Serotonergic Neurotransmission

Neurochemical, molecular, immunohistochemical and behavioral methods were used to examine the in vivo effects of the neuropeptide galanin on central 5-HT neurotransmission and on 5-HT1A receptor-mediated responses. Intraventricularly infused galanin caused a long-lasting and dose-dependent reduction of basal extracellular 5-HT levels in the ventral hippocampus of awake rats as measured by microdialysis. Infusion of galanin into the dorsal raphe nucleus (DRN), but not intrahippocampally, reduced 5-HT release. The effect of i.c.v. galanin on 5-HT release was blocked by the galanin receptor antagonist M35, acting most likely via galanin receptors at the level of the DRN. Galanin also reduced the levels of tryptophanhydroxylase mRNA in the DRN. Therefore, the effects of galanin on 5-HT1A receptor-mediated responses were further investigated. Surprisingly, galanin significantly attenuated the reduction of hippocampal 5-HT release induced by systemic injection of the 5-HT1A receptor agonist 8-OH-DPAT. Galanin also attenuated 8-OH-DPAT-induced hypothermia and locomotor activity in rats. These results indicate that galanin has important inhibitory actions on central 5-HT neurotransmission and on 5-HT1A receptor-mediated events.

Intraseptal muscarinic ligands and galanin: influence on hippocampal acetylcholine and cognition

The cholinergic neurons in the septohippocampal projection are implicated in hippocampal functions such as spatial learning and memory. The aim of this study was to examine how septohippocampal cholinergic transmission is modulated by muscarinic inputs and by the neuropeptide galanin, co-localized with acetylcholine (ACh) in septohippocampal cholinergic neurons, and how spatial learning assessed by the Morris water maze test is affected. Muscarinic inputs to the septal area are assumed to be excitatory, whereas galanin is hypothesized to inhibit septohippocampal cholinergic function. To test these hypotheses, compounds were microinjected into the medial septum and hippocampal ACh release was assessed by microdialysis probes in the ventral hippocampus of the rat. Blockade of septal muscarinic transmission by intraseptal scopolamine increased hippocampal ACh release suggesting that septal cholinergic neurons are under tonic inhibition. Stimulation of septal muscarinic receptors by carbachol also increased hippocampal ACh release. Despite this increase, both scopolamine and carbachol tended to impair hippocampus-dependent spatial learning. This finding also suggests a revision of the simplistic notion that an increase in hippocampal ACh may be facilitatory for learning and memory. Galanin infused into the medial septum enhanced hippocampal ACh release and facilitated spatial learning, suggesting that septal galanin, contrary to earlier claims, does not inhibit but excites septohippocampal cholinergic neurons. Galanin receptor stimulation combined with muscarinic blockade in the septal area resulted in an excessive increase of hippocampal ACh release combined with an impairment of spatial learning. This finding suggests that the level of muscarinic activity within the septal area may determine the effects of galanin on hippocampal cognitive functions. In summary, a limited range of cholinergic muscarinic transmission may contribute to optimal hippocampal function, a finding that has important implications for therapeutic approaches in the treatment of disorders of memory function.

Increased serotonin release in mice frontal cortex and hippocampus induced by acute physiological stressors

The effects of acute physiological stressors (5 s tail pinch, handling and forced swimming at +25 and +5 degrees C for 3 min each) on serotonin (5-HT) release in the mouse brain were investigated using in vivo microdialysis. The extracellular 5-HT levels were determined by a newly developed highly-sensitive and selective high-performance liquid chromatography method based on derivatization with benzylamine and fluorescence detection. The basal levels of 5-HT in 3 min microdialysates from the ventral hippocampus and frontal cortex were 0.68+/-0.21 and 0.75+/-0.28 fmol/6 microl (n=24), respectively. All three stressors caused an immediate, significant and reversible increase (handling: 150%; swimming: 240%) of extracellular 5-HT levels in both brain structures, suggesting a more dynamic role played by the serotonergic system in response to acute stress.

Transcription factor Nurr1 maintains fiber integrity and nuclear-encoded mitochondrial gene expression in dopamine neurons

Developmental transcription factors important in early neuron specification and differentiation often remain expressed in the adult brain. However, how these transcription factors function to mantain appropriate neuronal identities in adult neurons and how transcription factor dysregulation may contribute to disease remain largely unknown. The transcription factor Nurr1 has been associated with Parkinsons disease and is essential for the development of ventral midbrain dopamine (DA) neurons. We used conditional Nurr1 gene-targeted mice in which Nurr1 is ablated selectively in mature DA neurons by treatment with tamoxifen. We show that Nurr1 ablation results in a progressive pathology associated with reduced striatal DA, impaired motor behaviors, and dystrophic axons and dendrites. We used laser-microdissected DA neurons for RNA extraction and next-generation mRNA sequencing to identify Nurr1-regulated genes. This analysis revealed that Nurr1 functions mainly in transcriptional activation to regulate a battery of genes expressed in DA neurons. Importantly, nuclear-encoded mitochondrial genes were identified as the major functional category of Nurr1-regulated target genes. These studies indicate that Nurr1 has a key function in sustaining high respiratory function in these cells, and that Nurr1 ablation in mice recapitulates early features of Parkinsons disease.

Microdialysis in freely moving mice: determination of acetylcholine, serotonin and noradrenaline release in galanin transgenic mice

In the present study, we describe micro-surgical methods for simultaneous implantation of a microdialysis probe and an intraventricular injection cannula via their respective guide cannulas into the mouse brain. Basal and stimulated release of acetylcholine (ACh), serotonin (5-HT) and noradrenaline (NA) was determined in the ventral hippocampus of freely moving mice. NA and 5-HT were determined in one run by a newly developed HPLC method based on precolumn derivatization with benzylamine and fluorescence detection. The mice with a loss-of-function mutation of the galanin gene (KO) and the mice that over-expressed galanin (OE) were studied. No significant differences in basal, potassium-stimulated or scopolamine-induced extracellular ACh levels were observed in 4-month-old wild-type (WT) and KO mice. In the aged, 10-month-old animals, the basal extracellular ACh levels were significantly reduced in both WT and KO groups. Galanin (1 nmol i.c.v.) caused a significant reduction of basal extracellular NA by about 40% in both WT and galanin OE mice, however, in the latter group the effect was delayed by almost 2 h. A 10-min forced swimming stress caused a higher increase in release of NA and 5-HT in the OE group than in the corresponding WT mice. Finally, venlafaxin (10 mg/kg i.p.) increased extracellular NA to 400% of the control values in the CBA mice, but only to 250% in the C57BL mice. It is concluded that galanin may play an important role in the cholinergic mechanisms underlying cognitive disorders. Furthermore, modulation by galanin and by behavioral activation, of NA and 5-HT neurotransmission in galanin over-expressing mice indicates its possible role in the aetiology of mood disorders.

Simultaneous determination of norepinephrine, serotonin, and 5-hydroxyindole-3-acetic acid in microdialysis samples from rat brain by microbore column liquid chromatography with fluorescence detection following derivatization with benzylamine

A microbore column liquid chromatographic method for the simultaneous determination of norepinephrine (NE), serotonin (5-HT), and 5-hydroxyindole-3-acetic acid (5HIAA) in microdialysis samples from rat brain is described. The method is based on precolumn derivatization of NE, 5HT, and 5HIAA with benzylamine in the presence of potassium hexacyanoferrate(III) resulting in the corresponding highly fluorescent and stable benzoxazole derivatives. A 15-microl sample was mixed with 15 microl derivatization reagent solution containing 0.3M 3-cyclohexylaminopropanesulfonic acid buffer (pH 12.0), 0.5M benzylamine, 10mM potassium hexacyanoferrate(III), and methanol (1/1/1/12, v/v/v/v). The derivatization was carried out at 50 degrees C for 20 min. The benzylamine derivatives of NE, 5HT, and 5HIAA were separated on a reversed-phase column (100 x 1.0mm i.d., packed with C18 silica, 5 microm) within 30 min. The mobile phase consisted of 15 mM acetate buffer (pH 5.0) and acetonitrile (31%, v/v); the flow rate was 50 microl/min. The detection limits (signal-to-noise ratio of 3) for NE, 5HT, and 5HIAA in the injection volume of 20 microl were 90, 210, and 260 amol, respectively. Microdialysis samples were collected in 7.5-min intervals from the probes implanted in the hippocampus and prefrontal cortex of awake rats. The basal levels of NE, 5HT, and 5HIAA in the dialysates from the hippocampus were 4.2+/-0.5, 4.9+/-0.6, and 934.1 +/- 63.4 fmol/20 microl, and those from the prefrontal cortex were 6.0+/-1.2,5.51.3, and 669.1 +/- 96.0 fmol/20 microl (mean +/- SE, n=25), respectively. The NE and 5HT levels were altered by perfusion of high-potassium or low-calcium solution and following antidepressant drugs imipramine and desipramine. It is concluded that the new fluorescence derivatization method in combination with microbore column liquid chromatography allows the simultaneous determination of NE, 5HT, and 5HIAA in the microdialysis samples at higher sensitivity, providing easier maintenance in routine use than that achieved by high-performance liquid chromatographic methods with electrochemical detection.

The Ginkgo biloba extract EGb 761® and its main constituent flavonoids and ginkgolides increase extracellular dopamine levels in the rat prefrontal cortex

Background and purpose:  Experimental and clinical data suggest that extracts of Ginkgo biloba improve cognitive function. However, the neurochemical correlates of these effects are not yet fully clarified. The purpose of this study was to examine the effects of acute and repeated oral administration of the standardized extract EGb 761® on extracellular levels of dopamine, noradrenaline and serotonin (5‐HT), and the dopamine metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the prefrontal cortex (PFC) and striatum of conscious rats.

Galanin attenuates basal and antidepressant drug-induced increase of extracellular serotonin and noradrenaline levels in the rat hippocampus

Galanin is co-localized with classical neurotransmitters, such as acetylcholine, serotonin (5-HT) and noradrenaline (NA) in neurons or in brain regions implicated in cognitive and affective behaviour. In the present study, the effects of galanin on extracellular 5-HT and NA levels in the rat hippocampus were measured by in vivo microdialysis under basal conditions and following systemic administration of antidepressant drugs. Galanin (1.5 nmol i.c.v.) reduced basal 5-HT and NA levels to 65% and 86% of controls, respectively. Galanin (0.5 and 1.5 nmol i.c.v.) dose-dependently attenuated the elevation of 5-HT concentrations induced by imipramine and citalopram (10 mg/kg i.p., each) from 350% to 312% and from 230% to 160%, respectively. Galanin at 1.5 nmol transiently attenuated the effect of desipramine-induced (10 mg/kg i.p.) increase in extracellular NA levels from a maximal increase of 389-296% of the predrug levels. It is concluded that intraventricularly administered galanin attenuated both basal 5-HT and NA release and antidepressant drug-induced accumulation of extracellular 5-HT and NA levels most likely via a predominant inhibitory action on serotonergic and noradrenergic neurons in the raphe and locus coeruleus, respectively. These results further emphasize a possible role of galanin in regulation of 5-HT and NA neurotransmission in depressive states and during the course of antidepressant therapy.

Simultaneous determination of 5-hydroxyindoles and catechols by high-performance liquid chromatography with fluorescence detection following derivatization with benzylamine and 1,2-diphenylethylenediamine.

A highly selective and sensitive method for the simultaneous determination of 5-hydroxyindoles and catechols (serotonin, norepinephrine, dopamine and related compounds) by high-performance liquid chromatography with fluorescence detection is described. The method is based on the two-step precolumn derivatization of 5-hydroxyindoles and catechols with benzylamine (BA) and 1,2-diphenylethylenediamine (DPE), respectively, resulting in highly fluorescent and stable benzoxazole derivatives. The first derivatization with BA proceeds at room temperature (ca. 23 degrees C) for 2 min in a mixture of 0.3 M 3-cyclohexylamino-1-propanesulfonic acid buffer (pH 10.0) and methanol in the presence of potassium hexacyanoferrate(III). The subsequent second derivatization with DPE is carried out at 50 degrees C for 20 min in the presence of glycine. The resulting fluorescent derivatives of five 5-hydroxyindoles and seven catechols are separated on a reversed-phase column (150 x 1.5 mm I.D., packed with C18 silica, 5 microm) with isocratic elution using a mixture of acetonitrile-15 mM acetate buffer (pH 4.5) (34:66, v/v) containing 1 mM octanesulfonic acid sodium salt, and are detected spectrofluorimetrically at 480 nm with excitation at 345 nm. The detection limits (signal-to-noise ratio of 3) of the related compounds are 80 amol to 86 fmol for a 20-microl injection.