Serotonin Inhibits Acetylcholine Release from Rat Striatum Slices: Evidence for a Presynaptic Receptor-Mediated Effect

Rat brain striatum slices were incubated with [3H]choline, perfused with a physiological buffer, and stimulated by perfusion with a K+-enriched buffer for 2 min. The tritium overflow evoked by K+ was decreased by 5-hydroxytryptamine (serotonin, 5-HT) (maximal inhibition 10(-6) M). This effect of 5-HT was mimicked by several agonists (5-methoxytryptamine, N,N-dimethyl-tryptamine, bufotenin) and blocked by serotonergic antagonists (methiothepin, methysergide, cinanserin) but not by haloperidol; methiothepin and methysergide alone slightly increased the K+-evoked overflow of tritium (3H). Inhibition of the tritium release by 5-HT was not suppressed in the presence of tetrodotoxin (TTX) (10(-6) M). These results suggest that 5-HT tonically inhibits acetylcholine (ACh) release from striatal cholinergic neurons by acting on a presynaptic receptor localized on cholinergic terminals.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

Regional Alterations in Rat Brain Neurotransmitter Systems Following Chronic Lithium Treatment

Abstract: Chronic, but not acute, consumption of lithium leads to a significant decrease in serotonin and GABA receptor binding in selected regions of the rat brain, with no changes noted in P-adrenergic or cholinergic muscarinic receptor binding. In addition, the concentration of β-methoxytyramine, a dopamine metabolite, in the corpus striatum was increased in the animals treated chronically with lithium, suggesting a possible enhancement in dopamine release, or inhibition of uptake, in this brain area. In contrast, chronic consumption of rubidium had no effect on any of the parameters studied. The results suggest that lithium administration causes selective changes in brain neurotransmitter receptor systems and that the net result of these changes may be a decrease in GABAergic and serotoninergic activity. The fact that these alterktions are noted only after chronic administration suggests that they may be related to the therapeutic action of lithium in the prophylactic treatment of recurrent manic- depressive psychosis.     

The Amino Acid Content in Gamma-irradiated Rice†

High phosphate accumulating bacteria were isolated by autoradiography. One isoate, Arthrobacter globiformis PAB-6 accumulated phosphate intracellularly at 20% of dry cell mass in a simple synthetic medium. This amount was 3~7 times higher than type cultures examined. Almost no phosphate was released into the medium after cessation of growth. Fifty percent of total intracellular phosphate was fractionated as nucleic acids, while 20% each was recovered from cold PCA soluble fractions and polyphosphate fractions. The large content of nucleic acids in this bacterium appeared due to increased RNA content, specifically 4 S RNA fraction.     

Serotonin transporter gene,childhood emotional abuse and cognitive vulnerability to depression

Meta‐analyses evaluating the association between the serotonin transporter polymorphism (5‐HTTLPR) with neuroticism and depression diagnosis as phenotypes have been inconclusive. We examined a gene–environment interaction on a cognitive vulnerability marker of depression, cognitive reactivity (CR) to sad mood. A total of 250 university students of European ancestry were genotyped for the 5‐HTTLPR, including SNP rs25531, a polymorphism of the long allele. Association analysis was performed for neuroticism, CR and depression diagnosis (using a self‐report measure). As an environmental pathogen, self‐reported history of childhood emotional abuse was measured because of its strong relationship with depression. Participants with the homozygous low expressing genotype had high CR if they had experienced childhood emotional maltreatment but low CR if they did not have such experience. This interaction was strongest on the Rumination subscale of the CR measure. The interaction was not significant with neuroticism or depression diagnosis as outcome measures. Our results show that 5‐HTTLPR is related to cognitive vulnerability to depression. Our findings provide evidence for a differential susceptibility genotype rather than a vulnerability genotype, possibly because of the relatively low levels of abuse in our sample. The selection of phenotype and environmental contributor is pivotal in investigating gene–environment interactions in psychiatric disorders.     

Activation of Dopamine Receptors Does Not Affect Phosphoinositide Turnover in NCB-20 Cells

Dopamine inhibits and serotonin stimulates adenylate cyclase activity in a neuroblastoma X Chinese hamster brain explant cell line (NCB-20). The inhibition of cyclic AMP accumulation by dopamine was blocked by pretreatment of the cells with pertussis toxin. Carbachol and bradykinin stimulated the accumulation of water-soluble inositol phosphates whereas thyrotropin-releasing hormone, vasopressin, neurotensin, and phenylephrine were without effect. Dopamine and serotonin had no significant effect on carbachol-induced phosphoinositide hydrolysis or the levels of the parent lipids within the membrane. Forskolin induced a much larger stimulation of cyclic AMP than did serotonin, and caused an increase in the levels of phosphatidylinositol-4-phosphate and phosphatidyl inositol-4,5-bisphosphate in the cell membrane.     

A Ca2+-Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.     

N1-Methyl-2-125I-Lysergic Acid Diethylamide, a Preferred Ligand for In Vitro and In Vivo Characterization of Serotonin Receptors

Methylation of 2-125I-lysergic acid diethylamide (125I-LSD) at the N1 position produces a new derivative, N1-methyl-2-125I-lysergic acid diethylamide (125I-MIL), with improved selectivity and higher affinity for serotonin 5-HT2 receptors. In rat frontal cortex homogenates, specific binding of 125I-MIL represents 80-90% of total binding, and the apparent dissociation constant (KD) for serotonin 5-HT2 receptors is 0.14 nM (using 2 mg of tissue/ml). 125I-MIL also displays a high affinity for serotonin 5-HT1C receptors, with an apparent dissociation constant of 0.41 nM at this site. 125I-MIL exhibits at least 60-fold higher affinity for serotonin 5-HT2 receptors than for other classes of neurotransmitter receptors, with the dopamine D2 receptor as its most potent secondary binding site. Studies of the association and dissociation kinetics of 125I-MIL reveal a strong temperature dependence, with very slow association and dissociation rates at 0 degree C. Autoradiographic experiments confirm the improved specificity of 125I-MIL. Selective labeling of serotonin receptors was observed in all brain areas examined. In vivo binding studies in mice indicate that 125I-MIL is the best serotonin receptor label yet described, with the highest frontal cortex to cerebellum ratio of any serotonergic radioligand. 125I-MIL is a promising ligand for both in vitro and in vivo labeling of serotonin receptors in the mammalian brain.     

Morphological correlates of serotonin-neuropeptide Y interactions in the rat suprachiasmatic nucleus: Combined radioautographic and immunocytochemical data

Summary The morphological substrate of putative serotonin (5-HT)/neuropeptide Y (NPY) interactions in thé suprachiasmatic nucleus (SCN) was investigated by combined radioautography and immunocytochemistry after intraventricular administration of (³H)5-HT in the rat. In the ventral portion of the SCN, the distribution of (³H)5-HT uptake sites overlapped closely the NPY-immunoreactive terminals. Previous investigations have shown that the dense 5-HT and NPY innervations of the SCN originate in different structures, i.e., the midbrain raphe nuclei and the ventral lateral geniculate nucleus, respectively. Accordingly, in the present study, destruction of 5-HT afferents by 5,7-dihydroxytryptamine was not found to induce any modification in NPY staining and, in ultrastructural immuno-radioautographic preparations, two distinct pools of axonal varicosities could be identified. Both 5-HT and NPY terminals established morphologically defined synaptic junctions, sometimes on the same neuronal target. Some cases of direct axo-axonic appositions between the two types of terminals were also encountered. These data constitute additional criteria for characterizing the cytological basis of the multiple transmitter interactions presumably involved in the function of the SCN as a central regulator of circadian biological rhythms.     

Adrenergic nerves and 5-hydroxytryptamine-containing cells in the pulmonary vasculature of the aquatic file snake Acrochordus granulatus

Summary The adrenergic innervation of the pulmonary vasculature of the file snake Acrochordus granulatus was examined by use of glyoxylic acid-induced fluorescence. Perivascular plexuses of blue-green fluorescent nerves are observed around the common pulmonary artery, the anterior and posterior pulmonary arteries, the arterioles leading to the gas exchange capillaries of the lung, the venules draining the lung, and the anterior and posterior pulmonary veins. Adrenergic nerves are also associated with the visceral smooth muscle of the lung septa and other tissues. Thus, adrenergic control of pulmonary blood flow may occur either at the common pulmonary artery or more regionally within the lung. Regional control of blood flow in the elongate lung of this snake may be important in matching pulmonary perfusion with the distribution of respiratory gas. Glyoxylic acid-histochemistry and immunohistochemistry revealed that populations of cells located in the common pulmonary artery contain the indoleamine 5-hydroxy-tryptamine. Many of the cells are intimately associated with varicose blue-green fluorescent nerves. It is proposed that the 5-hydroxytryptamine-containing cells may be involved in intravascular chemoreception.