Vesicular monoamine transporters are known to transport monoamines from the cytoplasm into secretory vesicles. We have used homologous recombination to generate mutant mice lacking the vesicular monoamine transporter 2 (VMAT2), the predominant form expressed in the brain. Newborn homozygotes die within a few days after birth, manifesting severely impaired monoamine storage and vesicular release. In heterozygous adult mice, extracellular striatal dopamine levels, as well as K+- and amphetamine-evoked dopamine release, are diminished. The observed changes in presynaptic homeostasis are accompanied by a pronounced supersensitivity of the mice to the locomotor effects of the dopamine agonist apomorphine, the psychostimulants cocaine and amphetamine, and ethanol. Importantly, VMAT2 heterozygous mice do not develop further sensitization to repeated cocaine administration. These observations stress the importance of VMAT2 in the maintenance of presynaptic function and suggest that these mice may provide an animal model for delineating the mechanisms of vesicular release, monoamine function, and postsynaptic sensitization associated with drug abuse.
Glycogen synthase kinase-3beta (GSK-3beta) is thought to mediate morphological responses to a variety of extracellular signals. Surprisingly, we found no gross morphological deficits in nervous system development in GSK-3beta null mice. We therefore designed an shRNA that targeted both GSK-3 isoforms. Strong knockdown of both GSK-3alpha and beta markedly reduced axon growth in dissociated cultures and slice preparations. We then assessed the role of different GSK-3 substrates in regulating axon morphology. Elimination of activity toward primed substrates only using the GSK-3 R96A mutant was associated with a defect in axon polarity (axon branching) compared to an overall reduction in axon growth induced by a kinase-dead mutant. Consistent with this finding, moderate reduction of GSK-3 activity by pharmacological inhibitors induced axon branching and was associated primarily with effects on primed substrates. Our results suggest that GSK-3 is a downstream convergent point for many axon growth regulatory pathways and that differential regulation of primed versus all GSK-3 substrates is associated with a specific morphological outcome.
Brain dopamine (DA) systems are involved in the modulation of the sensorimotor gating phenomenon known as prepulse inhibition (PPI). The class of D2-like receptors, including the D2, D3, and D4 receptor subtypes, have all been implicated in the control of PPI via studies of DA agonists and antagonists in rats. Nevertheless, the functional relevance of each receptor subtype remains unclear because these ligands are not specific. To determine the relevance of each receptor subtype, we used genetically altered strains of "knock-out" mice lacking the DA D2, D3, or D4 receptors. We tested the effects of each knock-out on both the phenotypic expression of PPI and the disruption of PPI produced by the indirect DA agonist d-amphetamine (AMPH). No phenotypic differences in PPI were observed at baseline. AMPH significantly disrupted PPI in the D2 (+/+) mice but had no effect in the D2 (-/-) mice. After AMPH treatment, both DA D3 and D4 receptor (+/+) and (-/-) mice had significant disruptions in PPI. These findings indicate that the AMPH-induced disruption of PPI is mediated via the DA D2 receptor and not the D3 or D4 receptor subtypes. Uncovering the neural mechanisms involved in PPI will further our understanding of the substrates of sensorimotor gating and could lead to better therapeutics to treat gating disorders, such as schizophrenia.
Histamine is a neurotransmitter in the central The biogenic amine histamine is a neurotransmitter in the central nervous system and an important physiological modulator of gastric acid secretion, airway smooth-muscle tone, vasomotor control, inflammation, and allergic reactions (1-3). The formation of histamine from its precursor histidine is catalyzed by the enzyme L-histidine decarboxylase (HDC; L-histidine carboxy-lyase, EC 4.1.1.22). In rodents the enzyme is primarily localized in the brain (hypothalamic neurons and projections to other brain regions) (4), the glandular regions of the stomach (5), mast cells (6), and fetal liver (7). HDC has been purified from rat stomach (5) and fetal liver (7) and was shown to be a dimer consisting ofMr 54,000 subunits. Like other mammalian amino acid decarboxylases, the enzyme utilizes pyridoxal phosphate as a coenzyme. Although no primary amino acid sequence of mammalian or other eukaryotic HDC is known, immunological studies indicated that rodent HDC is closely related to L-dopa decarboxylase (DDC) (8). In this manuscript we present the amino acid sequence of rat HDC as deduced from the fetal liver cDNA11and demonstrate its close homology to DDC, but not to other characterized amino acid decarboxylases.
EXPERIMENTAL PROCEDURESPreparation and Screening of cDNA Library. Poly(A)+ RNA from fetal liver (16 days after conception) was prepared by guanidine thiocyanate extraction (9) and transcribed with avian myeloblastosis virus reverse transcriptase. The second strand was synthesized with RNase H and Escherichia coli DNA polymerase I (10). After treatment with T4 DNA polymerase to flush the ends, EcoRI linkers were applied by ligation and cleaved with EcoRI. Gel filtration (Sepharose CL-4B)-purified cDNA was ligated with dephosphorylated EcoRI-cleaved phage A gt11 DNA, and the recombinants were packaged in vitro (11). The unampliflied library was screened on E. coli Y1088, with [32P]DNA as previously described (12). Positive recombinants were plaque-purified, and the DNA inserts were isolated by electroelution.Transient Expression in Monkey Kidney Cells. The DNA inserts from isolates HDC-18 and HDC-21 were cloned into the expression vector pCMV5, a modified pCMV plasmid (13) containing an EcoRI cloning site (the modified plasmid was from David Russel and Matthew Lorence, University of Texas Southwestern Medical Center, Dallas). This plasmid vector contains the cytomegalovirus (CMV) promoterenhancer of the immediate early gene, the poly(A)+ additiontranscription terminator region of the human growth hormone gene, the simian virus 40 origin of replication, and a polylinker region for the insertion of cDNAs (13). Recombinants with the correct orientation for expression were identified by restriction endonuclease analysis, and the plasmid DNA (pCMVHDC-18 and pCMVHDC-21) was purified by CsCl/ethidium bromide centrifugation. For transfection, subconfluent COS 7 cells (106 cells per 100-mm dish) were transfected with 5 ug of pCMV DNA per dish by the DEAE-dextran method (14)...
The recent availability of mice lacking the neuronal form of the vesicular monoamine transporter 2 (VMAT2) affords the opportunity to study its roles in storage and release. Carbon fiber microelectrodes were used to measure individual secretory events of histamine and 5-hydroxytryptamine (5-HT) from VMAT2-expressing mast cells as a model system for quantal release. VMAT2 is indispensable for monoamine storage because mast cells from homozygous (VMAT2 ؊/؊ ) mice, while undergoing granule-cell fusion, do not release monoamines. Cells from heterozygous animals (VMAT2 ؉/؊ ) secrete lower amounts of monoamine per granule than cells from wild-type controls. Investigation of corelease of histamine and 5-HT from granules in VMAT2 ؉/؊ cells revealed 5-HT quantal size was reduced more than that of histamine. Thus, although vesicular transport is the limiting factor determining quantal size of 5-HT and histamine release, intragranular association with the heparin matrix also plays a significant role.
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