Diet-induced obesity causes hypothalamic neurochemistry alterations in Swiss mice.

The aim of this study was to assess inflammatory parameters, oxidative stress and energy metabolism in the hypothalamus of diet-induced obese mice. Male Swiss mice were divided into two study groups: control group and obese group. The animals in the control group were fed a diet with adequate amounts of macronutrients (normal-lipid diet), whereas the animals in the obese group were fed a high-fat diet to induce obesity. Obesity induction lasted 10 weeks, at the end of this period the disease model was validated in animals. The animals in the obese group had higher calorie consumption, higher body weight and higher weight of mesenteric fat compared to control group. Obesity showed an increase in levels of interleukin 1ß and decreased levels of interleukin 10 in the hypothalamus. Furthermore, increased lipid peroxidation and protein carbonylation, and decreased level of glutathione in the hypothalamus of obese animals. However, there was no statistically significant difference in the activity of antioxidant enzymes, superoxide dismutase and catalase. The obese group had lower activity of complex I, II and IV of the mitochondrial respiratory chain, as well as lower activity of creatine kinase in the hypothalamus as compared to the control group. Thus, the results from this study showed changes in inflammatory markers, and dysregulation of metabolic enzymes in the pathophysiology of obesity.

Neurochemistry in shiverer mouse depicted on MR spectroscopy.

PURPOSE: To evaluate the neurochemical changes associated with hypomyelination, especially to clarify whether increased total N-acetylaspartate (tNAA) with decreased choline (Cho) observed in the thalamus of msd mice with the plp1 mutation is a common finding for hypomyelinating disorders. MATERIALS AND METHODS: We performed magnetic resonance imaging (MRI) and proton MR spectroscopy ((1) H-MRS) of the thalamus and cortex of postnatal 12-week shiverer mice devoid of myelin basic protein (mbp), heterozygous and wild-type mice with a 7.0T magnet. Luxol Fast Blue staining and immunohistochemical analysis with anti-Mbp, Gfap, Olig2, and NeuN antibodies were also performed. RESULTS: In the thalamus, decreased Cho and normal tNAA were observed in shiverer mice. In the cortex, tNAA, Cho, and glutamate were decreased in shiverer mice. Histological and immunohistochemical analysis of shiverer mice brains revealed hypomyelination in the thalamus, white matter, and cortex; astrogliosis and an increased number of total oligodendrocytes in the white matter; and a decreased number of neurons in the cortex. CONCLUSION: The reduction of Cho on (1) H-MRS might be a common marker for hypomyelinating disorders. A normal tNAA level in the thalamus of shiverer mice might be explained by the presence of mature oligodendrocytes, which enable neuron-to-oligodendrocyte NAA transport or NAA catabolism.

Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

1-Methyl-1,2,3,4-tetrahydroisoquinoline antagonizes a rise in brain dopamine metabolism, glutamate release in frontal cortex and locomotor hyperactivity produced by MK-801 but not the disruptions of prepulse inhibition, and impairment of working memory in rat.

1-Methyl-1,2,3,4-tetrahydroisoquinoline (1MeTIQ) is an endogenous compound that is constantly present in the brain, and that exhibits neuroprotective activity. Our earlier study has suggested that 1MeTIQ may play a crucial physiological role in the mammalian brain as an endogenous regulator of dopaminergic activity. It is well known that central nervous system stimulants such as: amphetamine, cocaine, phencyclidine, and selective NMDA receptor antagonists, e.g., MK-801 produce neuropsychotoxicity (psychosis, addiction) that is indistinguishable from paranoid type schizophrenia. In rodents, phencyclidine and MK-801 are often used to evoke schizophrenia-like behavioral abnormalities which are inhibited by neuroleptics. The present study was designed to further investigate potential antipsychotic properties of 1MeTIQ by using both behavioral and neurochemical studies in the rat. We investigated the influence of 1MeTIQ (25 and 50 mg/kg ip) on locomotor hyperactivity, disruptions of prepulse inhibition (PPI), and working memory impairment induced by the NMDA receptor antagonist, MK-801 (0.2-0.3 mg/kg ip). In addition in the biochemical study, we analyzed the effect of 1MeTIQ on the changes in dopamine metabolism in different brain structures and in extraneuronal release of dopamine and glutamate in the rat frontal cortex, produced by MK-801. The concentration of dopamine (DA) and its metabolites: 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT), and homovanillic acid (HVA), as well as the extraneuronal concentration of dopamine and glutamate were established by HPLC. MK-801 (0.25 mg/kg ip) evoked significant disruptions of PPI and working memory impairment, and co-administration of 1MeTIQ at two investigated doses of 25 and 50 mg/kg ip did not antagonize these effects. On the other hand hyperactivity evoked by MK-801 as well as a rise in dopamine metabolism in specific brain structures and glutamate release in the frontal cortex was completely antagonized by pretreatment with 1MeTIQ. If the hyperlocomotion elicited by acutely administered MK-801 is a valid model of at least some aspects of schizophrenia, these results indicate that 1MeTIQ will show efficacy in treating this condition. In conclusions, the present study suggests that 1MeTIQ, an endogenous neuroprotective compound, exhibits also antipsychotic-like efficacy in some animal tests, and may be useful in clinical practice as a psychosis-attenuating drug in schizophrenic patients. However, 1MeTIQ did not attenuate sensorimotor gating deficit or working memory impairment evoked by MK-801 which may be served as a model of negative symptoms of schizophrenia.

Determination of high-affinity choline uptake (HACU) and choline acetyltransferase (ChAT) activity in the same population of cultured cells.

Cholinergic neurons are a major constituent of the mammalian central nervous system. Acetylcholine, the neurotransmitter used by cholinergic neurons, is synthesized from choline and acetyl CoA by the enzymatic action of choline acetyltransferase (ChAT). The transport of choline into the cholinergic neurons, which results in synthesis of ACh, is hemicholinium-sensitive and is referred to as high-affinity choline uptake (HACU). Thus, the formation of acetylcholine in cholinergic neurons largely depends on both the levels of choline being transported into the cells from the extracellular space and the activity of ChAT. Several methods were described previously to measure HACU and ChAT simultaneously in synaptosomes, but the same for cultured cells is lacking. We describe a procedure to measure HACU and ChAT at the same time in cultured cells by simple techniques employing radionuclides. In this procedure, we determined quantitatively hemicholinium-sensitive choline uptake and ChAT enzyme activity in a small number of differentiated human neuroblastoma (SK-N-SH) cells. We also determined the kinetics of choline uptake in the SK-N-SH cells. We believe that these simple methods can be used for neurochemical and drug discovery studies in several models of neurodegenerative disorders including Alzheimer's disease.

The brain selenoproteome: priorities in the hierarchy and different levels of selenium homeostasis in the brain of selenium-deficient rats.

The application of radionuclides for the localization of essential trace elements in vivo and the characterization of their binding proteins is a story of intermittently made improvements of the techniques used for their detection. In this study we present the use of neutron activation analysis and different autoradiographic imaging methods including real-time digital autoradiography to reveal new insights in the hierarchy of selenium homeostasis. Selenoproteins containing the essential trace element selenium play important roles in the CNS. Although the CNS does not show the highest selenium concentration in the case of selenium-sufficient supply in comparison with other organs, it shows a high priority for selenium uptake and retention in the case of dietary selenium deficiency. To characterize the hierarchy of selenium supply in the brain, in vivo radiotracer labeling with (75)Se in rats with different selenium status was combined with autoradiographic detection of (75)Se in brain tissue sections and (75)Se-labeled selenoproteins after protein separation by two-dimensional gel electrophoresis. This study demonstrates significant differences in the uptake of (75)Se into the brain of rats with different selenium status. A brain region-specific uptake pattern of the radiotracer (75)Se in selenium-deficient rats could be revealed and the CSF was identified as a key part of the brain selenium homeostasis.

Validation of a fluorescence-based high-throughput assay for the measurement of neurotransmitter transporter uptake activity.

Pre-synaptic dopamine, norepinephrine and serotonin transporters (DAT, NET and SERT) terminate synaptic catecholamine transmission through reuptake of released neurotransmitter. Common approaches for studying these transporters involve radiolabeled substrates or inhibitors which, however, have several limitations. In this study we have used a novel neurotransmitter transporter uptake assay kit. The assay employs a fluorescent substrate that mimics the biogenic amine neurotransmitters and is taken up by the cell through the specific transporters, resulting in increased fluorescence intensity. In order to validate the assay, a variety of reference and proprietary neurotransmitter transporter ligands from a number of chemical and pharmacological classes were tested. The ability of these compounds to inhibit the selective transporter-mediated uptake demonstrated a similar rank order of potency and IC(50) values close to those obtained in radiolabeled neurotransmitter uptake assays. The described assay enables monitoring of dynamic transport activity of DAT, NET and SERT and is amenable for high-throughput screening and compound characterization.

Measurement of GABA and glutamate in vivo levels with high sensitivity and frequency.

In the present protocol, we demonstrate a high-performance liquid chromatography (HPLC) system that enables detection of very low amounts of gamma-aminobutyric acid (GABA) (0.03 pmol) and glutamate (0.8 pmol). The HPLC system consists of two pumps, an electrochemical detector, a high-pressure six-way switching valve, a guard column, a microbore column, and a column oven. A microdialysis probe was implanted in the right parietal cortex in rats. Dialysates were collected every 5 min and were split into two equal aliquots for separate analysis of GABA and glutamate. After derivatization with o-phthalaldehyde (OPA), samples were isocratically separated and purified by the guard column. To make the peak of GABA or glutamate appear in an opportune place in a chromatogram, a six-way switching valve was used to control the eluate containing GABA or glutamate to be led to the microbore column and electrochemical detector. By the use of this system, decrease in extracellular concentration of GABA, which precedes the appearance of electrical discharge initiated by hyperbaric oxygen (HBO2) exposure, was detected by microdialysis at the time resolution of 5 min.

Effects of triiodothyronine and fluoxetine on 5-HT1A and 5-HT1B autoreceptor activity in rat brain: regional differences.

The thyroid hormone triiodothyronine (T3) augments and accelerates the effects of antidepressant drugs. Although the majority of studies showing this have used tricyclics, a few studies have shown similar effects with the selective serotonin re-uptake inhibitor (SSRI) fluoxetine. In this study we investigated the effects of fluoxetine (5 mg/kg), T3 (20 microg/kg) and the combination of these drugs, each administered daily for 7 days, on serotonergic function in the rat brain, using in vivo microdialysis. Fluoxetine alone induced a trend towards desensitization of 5-HT1A autoreceptors as shown by a reduction in the effect of 8-OH-DPAT to lower 5-HT levels in frontal cortex, and desensitized 5-HT1B autoreceptors in frontal cortex. The combination of fluoxetine and T3 induced desensitization of 5-HT1B autoreceptors in hypothalamus. Since there is evidence linking hypothalamic function and depression, we suggest that this effect may partly account for the therapeutic efficacy of the combination of an SSRI and T3.

Identification of neurite extension inducing peptides by means of soluble combinatorial peptide libraries.

To identify hexapeptides capable of inducing neurite outgrowth, we used three groups of soluble combinatorial peptide libraries each consisting of 100 mixtures of hexapeptides (each mixture consisting of 10,000 individual peptides) with partially predetermined sequences (in two out of six amino acid positions). Using this approach a number of neuritogenic peptides were identified. Three selected peptides, QSGKKF, QSGPLA and QSGKQG, were found to induce neurite outgrowth from primary hippocampal neurons with potency comparable to that of growth factors. None of the peptides protected cerebellar granule neurons from cell death induced by withdrawal of potassium chloride. The approach described here suggests the feasibility to use combinatorial peptide libraries in order to identify compounds capable of modulating a specific functional response in the nervous system, without prior knowledge of a molecular target.

Human thalamus: neurochemical mapping of inferior pulvinar complex.

The primate pulvinar connects with the entire array of known visual areas and is postulated to play a role in selective visual attention. Recently, five separate neurochemical subdivisions of a region termed the inferior pulvinar (PI) complex were identified in monkeys. In the present study, similar histochemical procedures were applied to map the extent of the PI complex in humans. Acetylcholinesterase histochemistry and cytochrome oxidase staining demarcated four histochemical zones in human pulvinar, corresponding to the medial, central, lateral and lateral-shell (PI(M), PI(C), PI(L), and PI(L-S)) divisions of the PI complex in monkeys.

Neurochemical identification of A13 dopaminergic neuronal projections from the medial zona incerta to the horizontal limb of the diagonal band of Broca and the central nucleus of the amygdala.

Studies utilizing fluorescent histochemical techniques first revealed that A13 dopaminergic (DA) perikarya located in medial zona incerta (MZI) project to various regions within the hypothalamus; accordingly, these DA neurons were designated the 'incertohypothalamic' DA neuronal system. More recently, it has been shown that the anterograde neuronal tract tracer Phaseolus vulgaris leucoagglutinin, after injection into MZI, is identified in nerve terminals outside of the hypothalamus; for example, in horizontal limb of the diagonal band of Broca (HDB) and central nucleus of the amygdala (cAMY). The purpose of the present study was to determine, using neurochemical techniques, if A13 DA neurons project to the HDB and cAMY. Concentrations of dopamine and one of its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) were determined in HDB and cAMY following: (1) electrical stimulation of MZI, (2) electrolytic lesion or knife ablation of MZI, and (3) administration of gamma-hydroxybutyric acid (GHBA) into MZI. For comparison, similar measurements were made in nucleus accumbens (N. Acc.), a terminal region of A10 DA neurons in the ventral tegmental area (VTA). Electrical stimulation of MZI increased DOPAC concentrations in HDB and cAMY, whereas electrolytic or ablative lesions of MZI decreased dopamine concentrations in both of these regions. By contrast, neither stimulation nor lesion of MZI had any effect on DOPAC or dopamine concentrations in N. Acc. Intracerebral injection of GHBA into MZI increased dopamine concentrations in MZI and HDB, but not in cAMY or N. Acc. Intracerebral administration of GHBA into VTA increased dopamine concentrations in HDB and N. Acc., but not in MZI or cAMY.(ABSTRACT TRUNCATED AT 250 WORDS)