Electrophysiological characterization of the effect of long-term duloxetine administration on the rat serotonergic and noradrenergic systems

3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferases (KdtA) are multifunctional glycosyltransferases with primary structures of low similarity. Totally degenerated primers were deduced from two stretches of identical amino acids between known KdtA sequences and used to amplify by PCR a kdtA-specific fragment from Acinetobacter baumannii ATCC 15308 DNA which was then applied as a probe for the cloning and sequencing of the complete Kdo transferase gene. With conserved PCR primers for this structural gene from A. baumannii ATCC 15308, also kdtA genes of A. baumannii ATCC 19606 and A. haemolyticus ATCC 17906 were obtained, cloned from the chromosome and sequenced. The genes coded for proteins with similarities to known Kdo transferases. Within the genus Acinetobacter, the identity and similarity of the deduced amino acid sequences were 71% and 84.5%, respectively. The kdtA sequences of both A. baumannii strains were identical and possessed a TTG start codon, whereas ATG was found in the case of A. haemolyticus. The genes from Acinetobacter and kdtA from Escherichia coli K-12 were expressed in the Gram-positive bacterium Corynebacterium glutamicum. In vitro tests confirmed the function of the gene products as Kdo transferases, which transferred mainly two Kdo residues to a synthetic lipid A precursor of E. coli. Also, no differences between the cloned kdtA genes from A. baumanniii, A. haemnolyticus and E. coli were observed when tetraacyl or hexaacyl lipid A were tested, since all transferases acted more efficiently on the former. With limiting amounts of acceptor, all Kdo transferases were able to transfer a third Kdo residue with varying efficiency.     

Astroglial and vascular interactions of noradrenaline terminals in the rat cerebral cortex

Noradrenaline (NA) has been shown to influence astrocytic and vascular functions related to brain homeostasis, metabolism, local blood flow, and blood-brain barrier permeability. In the current study, we investigate the possible associations that exist between NA-immunoreactive nerve terminals and astrocytes and intraparenchymal blood vessels in the rat frontoparietal cortex, both at the light and electron microscopic levels. As a second step, we sought to determine whether the NA innervation around intracortical microvessels arises from peripheral or central structures by means of injections of N-(2-chloroethyl-N-ethyl-2-bromobenzylamine) (DSP-4), a neurotoxin that specifically destroys NA neurons from the locus ceruleus. At the light microscopic level, 6.8% of all NA-immunoreactive nerve terminals in the frontoparietal cortex were associated with vascular walls, and this perivascular noradrenergic input, together with that of the cerebral cortex, almost completely disappeared after DSP-4 administration. When analyzed at the ultrastructural level in control rats, NA terminals in the neuropil had a mean surface area of 0.53 +/- 0.03 micron2 and were rarely junctional (synaptic incidence close to 7%). Perivascular terminals (located within a 3-micron perimeter from the vessel basal lamina) counted at the electron microscopic level represented 8.8% of the total NA terminals in the cortical tissue. They were smaller (0.29 +/- 0.01 micron2, P < 0.05) than their neuronal counterparts and were located, on average, 1.34 +/- 0.08 microns away from intracortical blood vessels, which consisted mostly of capillaries (65%). None of the perivascular NA terminals engaged in junctional contacts with surrounding neuronal or vascular elements. The primary targets of both neuronal and perivascular NA nerve terminals consisted of dendrites, nerve terminals, astrocytes, and axons, whereas in the immediate vicinity (0.25 micron or less) of the microvessels, astrocytic processes represented the major target. The results of the current study show that penetrating arteries and intracortical microvessels receive a central NA input, albeit parasynaptic in its interaction, originating from the locus ceruleus. Particularly, they point to frequent appositions between both neuronal and perivascular NA terminals and astroglial cells and their processes. Such NA neuronal-glial and neuronal-glial-vascular associations could be of significance in the regulation of local metabolic and vascular functions under normal and pathologic situations.     

Quantitative data on serotonin nerve terminals in adult rat neocortex

Serotonin (5-HT) nerve terminals, specifically labeled with [3H]5-HT have been counted in light microscope radioautographs from the fronto-parietal neocortex of adult rats, following prolonged superfusions with relatively high concentrations of tracer (10(-5) M or 10(-4) M), and after 15 or 30 days of radioautographic exposure. Comparative analysis of the results indicated that all 5-HT varicosities did not possess the same capacity to accumulate the exogenous amine. Nevertheless, superfusions with 10(-4) M[3H]5-HT provided a sufficient loading of 5-HT boutons to allow their complete detection within any given layer of the neocortex. The labeled varicosities were found to be present within all cortical layers, except layer VI. Their total number was extrapolated to be approximately 1 X 10(6)/cu.mm of cortex. This figure represented a mean incidence of one 5-HT nerve terminal/850-1400 cortical synapses (0.07-0.12%). The intralaminar density of 5-HT innervation increased progressively from layer V to layer I, in a distribution pattern suggestive of unspecific afferents. Based on endogenous 5-HT levels of 346 +/- 32 ng/g for the fronto-parietal neocortex, the mean 5-HT content per varicosity could be estimated to be 0.35 X 10(-3) pg, and the concentration 1900 ng/g wet weight of terminal or 0.2%. In view of their particular ultrastructural features7, ubiquitous repartition and endogenous amine content, 5-HT nerve endings may be considered as capable of exerting a rather widespread influence in the neocortex.     

Antagonism of central CRF systems mediates stress-induced changes in noradrenaline and serotonin turnover in rat brain regions

OBJECTIVE: We investigated changes in motor evoked potentials (MEPs) to explain why mental practice can improve motor performance. METHODS: MEPs were recorded from right and left first dorsal interosseous (FDI) muscles of 9 normal, right-handed subjects during different motor images of index finger movement: (1) rest, (2) flexion, (3) abduction, (4) extension. A paired t test was used to compare differences of stimulus intensities and MEP amplitudes among conditions. RESULTS: MEP amplitudes significantly increased in both FDI muscles during motor images of flexion and abduction but not of extension. Moreover, MEP amplitudes were larger in flexion than in abduction. These differences were proportional to the amount of real EMG discharge of FDI muscle in the selected direction of index finger movement. With regard to right-left differences, MEP amplitudes in the right FDI muscle were larger than those in the left. CONCLUSIONS: The primary motor cortex plays a role in the mental representation of motor acts. Furthermore, the amount of corticomotoneuronal cell activity is affected by the different motor images utilizing the same muscle. Right-left difference of MEP amplitude supports the view of left-hemisphere dominance for motor programming as an aspect of normal brain function among right-handers.     

Effects of acute and repeated administration of citalopram on extracellular levels of serotonin in rat brain

The effects of acute (2 days) and repeated (21 days) administration (50 mg/kg in the diet) of the selective serotonin (5-HT, 5-hydroxytryptamine) reuptake inhibitor, citalopram, on extracellular levels of 5-HT and their modulation by terminal autoreceptors in the hypothalamus of freely moving rats were compared in vivo by microdialysis. When studied without washout, extracellular levels of 5-HT were increased by both acute and repeated citalopram administration. In rats treated repeatedly, extracellular 5-HT levels were 43% (but not significantly) greater than in those treated acutely. Extracellular levels of 5-HT in control and citalopram-treated rats were similar when measured after 24 h washout. The enhancing effect of non-selective serotonergic autoreceptor antagonists, methiothepin (100 microM) or 1-(1-naphthyl)piperazine (NP) (10 microM), administered through the microdialysis probe, after 24 h washout, was similar in both control and chronically treated groups. These results suggest that repeated administration of citalopram followed by a washout of 24 h does not lead to desensitization of the terminal autoreceptor as measured in vivo in contrast to the effects we have shown previously in vitro. In rats treated chronically with citalopram without washout, methiothepin had a greater maximal effect on 5-HT outflow in comparison to rats receiving acute citalopram treatment. This finding suggests that a 5-HT autoreceptor antagonist or a combination of such a drug with a 5-HT uptake inhibitor would produce a greater increase of extracellular levels of 5-HT in hyposerotonergic states such as depression.     

Effect of acetylsalicylic acid on formalin test and on serotonin system in the rat brain

1. Acetylsalicylic acid (ASA; 400 mg/kg, i.p.) increased serotonin (5-HT) content in rat brain but did not modify the number or the affinity of 5-HT1A receptors in the pons and the cerebral cortex, whereas the number of cortical 5-HT2 receptors decreased significantly. 2. Pretreatment with parachlorophenylaline (100 mg/kg/day for 4 days) depleted 5-HT brain content but modified neither the serum levels of salicylates nor the 5-HT2 cortical receptor characteristics, and it abolished the antinociceptive effect of ASA, 400 mg/kg, in the first phase of the formalin test. 3. These data support the involvement of the central serotonergic system in the antinociceptive activity of ASA.     

Prevention by calcium administration of reserpine action on rat brain noradrenaline stores: a reappraisal

The conditions under which pretreatment with a calcium salt may prevent the action of reserpine on brain noradrenaline stores in the rat were investigated. The results show that only after subcutaneous administration of reserpine in the same site as a previous CaCl2 injection, was the action of reserpine prevented and reduced levels of this drug were found in the brain. Conversely, the depletion of encephalic noradrenaline following reserpine, as well as the reserpine brain concentration, were not affected by subcutaneously administered calcium chloride, when reserpine was administered either intravenously, or subcutaneously in a site different from that selected for pretreatment with the calcium salt. In essence calcium chloride, a well known irritant, acts accordingly at the site of subcutaneous administration, thus limiting by a non specific mechanism the absorption of reserpine. Under similar conditions, in fact, the absorption of a different drug, i.e. harmaline, was likewise altered. In view of these findings the significance of some studies on calcium-reserpine interaction appearing in the literature requires a reappraisal.     

Effect of repeated immobilization on serotonin metabolism in different rat brain areas and on serum corticosterone

The effect of daily repeated 10 min immobilization on the serotoninergic neurotransmission and serum corticosterone levels was studied. Male Lewis rats were immobilized for a 10 min period daily once or on 5 consecutive days. Serotoninergic neurotransmission was followed using differential in vivo pulse voltammetry with carbon fibre electrodes measuring extracellular 5-hydroxyindoleacetic acid (5-HIAA) levels. Recordings were performed in brain areas involved in the control of behaviour, mood, and stress response such as the frontal cortex, the hippocampal CA-3 and dentate gyrus, the striatum, and the raphe nuclei dorsalis (NRD) and medialis (MRN). The first immobilization resulted in an increase of the extracellular 5-HIAA levels in all areas under study, except the striatum where no reaction was observed. The major effect was recorded in the frontal cortex, showing an increase of about 400% as compared to control, which lasted for 3h after the end of the immobilization period. Beginning on day 2 in all areas, except the striatum, a consecutive habituation to the stressor seemed to occur, since the stress-induced increase in the voltammetric signal was found to be reduced after consecutive immobilization. Serum corticosterone levels were measured directly after a single and after 5 daily immobilization periods. After single immobilization the serum corticosterone level was found to be about 270 ng/ml. After the 5th immobilization about 300 ng/ml were detected. These differences were not found to be significant. In summary, our data indicate that the serotonin metabolism shows habituation in nearly all brain areas after repeated immobilization, though the corticosterone level at the end of the immobilization period was comparable after single and repeated immobilization.     

Effect of variations in adrenocortical function on dopamine beta-hydroxylase and norepinephrine in the brain of the rat

The effect of adrenalectomy and corticosterone treatment on dopamine beta-hydroxylase (DBH) activity, catecholamine content and norepinephrine formation and metabolism were studied in the hypothalamus and other parts of the brain of male rats. Two days after adrenalectomy, there was a decrease in DBH activity in the hypothalamus and the brain stem but no change in norepinephrine or dopamine content. Conversion of intraventricularly administered tritiated dopamine to tritiated norepinephrine was slightly increased and norepinephrine was metabolized at a more rapid rate than normal. Corticosterone in a dose of 100 mg/kg increased DBH activity but decreased hypothalamic norepinephrine and copamine content. In adrenalectomized rats, smaller, more physiological doses of corticosterone did not change DBH activity or catecholamine content. The fact that norepinephrine formation and metabolism were increased at the same time that DBH activity in vitro was decreased suggests that DBH is not rate-limiting in adrenergic neurons in the hypothalamus, or that a change in the in vitro activity of the enzyme was not accompanied by a parallel change in its activity in vivo.     

Effect of chronic administration of fenfluramine and quipazine on body weight gain after ovariectomy and on brain serotonin receptor binding.

In Exp I, chronic fenfluramine administration prevented excess weight gain following ovariectomy in 24 female Long-Evans rats but did not alter weight gain in 27 sham-operated controls. Chronic quipazine treatment had no long-term effect on body weight. Fenfluramine treatment for 28 days produced a 13% decrease in [–3H]-serotonin binding to hypothalamic membranes and no change in [–3H]-spiroperidol binding to striatum. In Exp II with 10 female Sprague-Dawley rats, chronic fenfluramine had no effect on [–3H]-serotonin binding to diencephalon. Results are discussed in terms of central and peripheral mechanisms of action and tolerance to fenfluramine. (10 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

P2-receptor-mediated inhibition of serotonin release in the rat brain cortex

The possibility of a P2-receptor-mediated modulation of the release of serotonin in the rat brain cortex was investigated in occipito-parietal slices preincubated with [3H]serotonin and then superfused and stimulated electrically (10 pulses, 1 Hz). Adenosine receptor agonists decreased the stimulation-evoked overflow of tritium at best slightly; the selective A1 agonist N6-cyclopentyl-adenosine caused no change. Several nucleotides had more marked effects: ATP (3-1000 microM), adenosine-5'-O-(3-thiotriphosphate) (3-300 microM) and P1,P5-di(adenosine-5')-pentaphosphate (3-300 microM) decreased the evoked overflow by up to ca 35%. AMP, alpha,beta-methylene-ATP and UTP produced smaller decreases and 2-methylthio-ATP and UMP caused no change. The inhibition by ATP was attenuated both by the P1-receptor antagonist 8-(p-sulphophenyl)-theophylline (100 microM) and by the P2-receptor antagonist suramin (300 microM) but was not changed by indomethacin (10 microM) and NG-nitro-L-arginine (10 microM). We conclude that the release of serotonin in the rat brain cortex is inhibited through presynaptic P1-receptors (which are not A1) as well as P2-receptors. Inhibition of release via P2-receptors has been previously shown for noradrenaline (brain cortex and hippocampus) and dopamine (neostriatum) and, hence, may be widespread. Differences between transmitter systems exist, however, in the degree of their sensitivity to presynaptic P2-receptor-mediated modulation.     

Direct observation of serotonin 5-HT3 receptor-induced increases in calcium levels in individual brain nerve terminals

Confocal microscopy was used to assess internal calcium level changes in response to presynaptic receptor activation in individual, isolated nerve terminals (synaptosomes) from rat corpus striatum, focusing, in particular, on the serotonin 5-HT3 receptor, a ligand-gated ion channel. The 5-HT3 receptor agonist-induced calcium level changes in individual synaptosomes were compared with responses evoked by K+ depolarization. Using the fluorescent dye fluo-3 to measure relative changes in internal free Ca2+ concentration ([Ca2+]i), K+-induced depolarization resulted in variable but rapid increases in apparent [Ca2+]i among the individual terminals, with some synaptosomes displaying large transient [Ca2+]i peaks of varying size (two- to 12-fold over basal levels) followed by an apparent plateau phase, whereas others displayed only a rise to a sustained plateau level of [Ca2+]i (two- to 2.5-fold over basal levels). Agonist activation of 5-HT3 receptors induced slow increases in [Ca2+]i (rise time, 15-20 s) in a subset (approximately 5%) of corpus striatal synaptosomes, with the increases (averaging 2.2-fold over basal) being dependent on Ca2+ entry and inhibited by millimolar external Mg2+. We conclude that significant increases in brain nerve terminal Ca2+, rivaling that found in response to excitation by depolarization but having distinct kinetic properties, can therefore result from the activation of presynaptic ligand-gated ion channels.     

Effect of alpha-methyldopa and alpha-methyl-m-tyrosine on the amount of noradrenaline in the heart and brain of irradiated rats

Effect of alpha-methyldopa and alpha-methyl-m-tyrosine on the amount of noradrenaline in the heart and brain of rats irradiated with 850 R was investigated. It was established that the application of alpha-methyldopa to the animals irradiated with 650 R caused significant decrease of noradrenaline stores in the heart and brain. Meanwhile, when alpha-methyldopa was applied to the animals irradiated with 850 R, there was no further decreasing of noradrenaline. The different effects may be tentatively explained: in animals irradiated with 650 R the processes of biosynthesis are still going on, while after the dose of 850 R, biosynthesis of catecholamine is badly damaged and the further inhibition of decarboxilase is probably impossible. Alpha-methyl-m-tyrosine caused, also, the strong discharge of noradrenaline storage in the heart and brain of irradiated animals. It is interesting to note there is no significant difference in inhibitory effect on animals irradiated with 650 and 850 R, respectively.     

Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.     

Immunocytochemical localization and description of neurons expressing serotonin2 receptors in the rat brain

Serotonin2 receptors have been implicated in a variety of behavioral and physiological processes, as well as a number of neuropsychiatric disorders. To specify the brain regions and specific cell types possessing serotonin2 receptors, we conducted an immunocytochemical study of the rat brain using a polyclonal serotonin2 receptor antibody. Perfusion-fixed rat brain sections were processed for immunocytochemistry and reactivity was visualized using an immunoperoxidase reaction. Numerous small, round neurons were heavily labeled in the granular and periglomerular regions of the olfactory bulb. Heavy labeling of medium-sized multipolar and bipolar neurons was also seen in olfactory regions of the ventral forebrain, including the anterior olfactory nucleus and olfactory tubercle. Other regions of the basal forebrain exhibiting high levels of immunoreactivity were the nucleus accumbens, ventral pallidum, Islands of Calleja, fundus striatum and endopyriform nucleus. Immunoreactive neurons were also seen in the lateral amygdala. A dense band of small, round cells was stained in layer 2 of pyriform cortex. In neocortex, a very sparse and even distribution of bipolar and multipolar neurons was seen throughout layers II-VI. A much more faintly labeled population of oval cells was observed in the deep layer of retrosplenial and posterior cingulate cortex, and in the granular layer of somatosensory frontoparietal cortex. A moderate number of medium bipolar and multipolar cells were scattered throughout the neostriatum, and a moderate number of pyramidal and pyramidal-like cells were seen in the CA fields of the hippocampus. Diencephalic areas showing immunolabeling included the medial habenula and anterior pretectal nucleus, with less labeling in the ventral lateral geniculate. In the hindbrain, two dense populations of large multipolar cells were heavily labeled in the pedunculopontine and laterodorsal tegmental nuclei, with lesser labeling in the periaqueductal gray, superior colliculus, spinal trigeminal nucleus and nucleus of the solitary tract. Based on the distribution, localization and morphology of immunoreactive neurons in these regions, we hypothesize that subpopulations of serotonin2 containing cells may be GABAergic interneurons or cholinergic neurons. Further, the observed distribution suggests that the physiological effects of serotonin acting through serotonin2 receptors are mediated by a relatively small number of cells in the brain. These observations may have strong functional implications for the pharmacological treatment of certain neuropsychiatric disorders.     

The relationship between the degree of neurodegeneration of rat brain 5-HT nerve terminals and the dose and frequency of administration of MDMA ('ecstasy')

The effect of varying the dose and frequency of administration of 3,4-methylenedioxymethamphetamine (MDMA or 'ecstasy') on both the acute hyperthermic response and the long term neurodegeneration of 5-hydroxytryptamine (5-HT) nerve terminals in the brain has been studied in Dark Agouti rats. A single injection (4-15 mg/kg i.p.) of MDMA produced immediate dose-related hyperthermia and a dose-related decrease in 5-HT, 5-hydroxyindoleacetic acid (5-HIAA) and [3H]paroxetine binding in regions of the brain 7 days later, with a dose of 4 mg/kg having no degenerative effect. This dose was also without effect when given once daily for 4 days, but produced a marked loss of [3H]paroxetine binding and indole concentration ( approximately 55%) when given twice daily for 4 days. When a dose of 4 mg/kg was given twice weekly for 8 weeks it had no effect on these serotoninergic markers, despite a clear anorectic effect of the drug being seen. These data demonstrate that MDMA-induced neurodegeneration is related to both the dose and frequency of administration and indicate that damage to 5-HT neurones can occur in the absence of a hyperthermic response to the drug. We suggest that damage occurs when endogenous free radical scavenging mechanisms become overwhelmed or exhausted.     

Effects of short-and long-term administration of tricyclic antidepressants and lithium on norepinephrine turnover in brain

The findings summarized in this paper show that norepinephrine turnover in brain is decreased after acute administration of imipramine or desmethylimipramine but tends to increase during chronic administration of these tricyclic antidepressants. Similarly, it appears that there also may be important differences between the effects of acute and chronic administration of lithium salts on norepinephrine turnover in the central nervous system. Such changes in norepinephrine turnover that develop gradually over the course of long-term drug administration may help to explain the need for chronic administration of tricyclic antidepressants or lithium salts in the treatment of patients with affective disorders.