Extremely high titre polyclonal antisera against small neurotransmitter molecules: rapid production, characterisation and use in lightand electron-microscopic immunocytochemistry

Pow, David V.; Crook, Denise K.

doi:10.1016/s0165-0270(05)80007-x

Cited by 132 publications

(111 citation statements)

References 13 publications

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“…This solution was centrifuged (20,000 ϫ g; 15 min), and the reddish pellet was resuspended in a solution containing dialyzed GST-VGLUT3 fusion protein (2 mg/ml), mixed with Freund's adjuvant, and used as the immunogen in guinea pigs. A similar immunogold immunization protocol has successfully produced extremely high titer polyclonal antibodies (30).…”

Section: Methodsmentioning

confidence: 99%

Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons

Schäfer¹,

Varoqui²,

Defamie³

et al. 2002

Journal of Biological Chemistry

371

392

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We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H ؉ -dependent, was stimulated by Cl ؊ ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.

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Section: Methodsmentioning

confidence: 99%

Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons

Schäfer¹,

Varoqui²,

Defamie³

et al. 2002

Journal of Biological Chemistry

371

392

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“…Citrulline was coupled to BSA with glutaraldehyde and then reduced with NaBH 4 (21). After extensive dialysis against water, the conjugate was adsorbed to freshly prepared 45-nm colloidal gold particles (22). A rabbit was immunized intradermally every 3 weeks with the BSA conjugate alone and i.v.…”

Section: Methodsmentioning

confidence: 99%

Neuronal nitric oxide synthase alternatively spliced forms: Prominent functional localizations in the brain

Eliasson

Blackshaw

Schell

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

265

220

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Neuronal nitric-oxide synthase (nNOS) is subject to alternative splicing. In mice with targeted deletions of exon 2 (nNOS ⌬͞⌬ ), two alternatively spliced forms, nNOS␤ and ␥, which lack exon 2, have been described. We have compared localizations of native nNOS␣ and nNOS␤ and ␥ by in situ hybridization and immunohistochemistry in wild-type and nNOS ⌬͞⌬ mice. To assess nNOS catalytic activity in intact animals we localized citrulline, which is formed stoichiometrically with NO, by immunohistochemistry. nNOS␤ is prominent in several brain regions of wild-type animals and shows 2-to 3-fold up-regulation in the cortex and striatum of nNOS ⌬͞⌬ animals. The persistence of much nNOS mRNA and protein, and distinct citrulline immunoreactivity (cit-IR) in the ventral cochlear nuclei and some cit-IR in the striatum and lateral tegmental nuclei, indicate that nNOS␤ is a major functional form of the enzyme in these regions. Thus, nNOS␤, and possibly other uncharacterized splice forms, appear to be important physiological sources of NO in discrete brain regions and may account for the relatively modest level of impairment in nNOS ⌬͞⌬ animals.Nitric oxide (NO), formed by neuronal nitric-oxide synthase (nNOS) (1), has major signaling functions in the central and peripheral nervous system (2, 3). Alternative splicing of the nNOS gene is prominent with more than 10 differently spliced nNOS transcripts reported (4-10). The principal, exon 2-containing, form of nNOS (nNOS␣) accounts for the great majority of catalytic activity in the brain, as mice with targeted deletions of exon 2 (nNOS ⌬͞⌬ ) display an Ϸ95% reduction in NOS catalytic activity (11). Though NO has been implicated in development (12) and synaptic plasticity (13), these mice appear grossly normal, lack obvious histopathological abnormalities in the central nervous system, and reproduce effectively (11), suggesting that the residual NOS activity might protect the nNOS ⌬͞⌬ mutant mice from serious pathology. Two alternatively spliced forms of nNOS, ␤ and ␥, that lack exon 2 and thus persist in the nNOS ⌬͞⌬ mice, have a regional distribution that parallels the pattern of the residual activity (4).The human nNOS gene contains 29 exons of which all but exon 1 are translated to generate nNOS␣, a 150-kDa protein (1, 8). nNOS␤ and ␥, as described in mice, are generated by alternative splicing that skips exon 2 and employs different first exons, 1a and 1b, respectively (4) (Fig. 1). Translation of nNOS␤ is initiated at a CTG-initiation codon within exon 1a generating a 136-kDa NOS protein with six unique N-terminal amino acids. Translation of nNOS␥ is initiated at an ATGinitiation codon within exon 5 generating a truncated, 125-kDa form of nNOS␣. Both nNOS␤ and nNOS␥ lack the PSD-95͞ discs large͞ZO-1 homology domain (PDZ) domain that is localized within exon 2. This domain, also referred to as DHR or GLGF, mediates association of nNOS␣ with postsynaptic density protein 95 ref. 4). PSD-95 appears to anchor nNOS␣ to neuronal membranes in the vicinity of the Nmethyl-D-as...

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“…Immunocytochemical detection of endogenous glutamate was carried out as previously described using well-characterized antibodies against glutamate, [25][26][27] the tissues being fixed with 2.5% glutaraldehyde in 0.1 mol l 21 sodium cacodylate buffer and then processed for embedding in araldite resin. Semithin (0.5 mm) sections were subsequently cut and immunostained as previously described.…”

Section: Glutamate Labelingmentioning

confidence: 99%

Expression of multiple glutamate transporter splice variants in the rodent testis

Lee¹,

Anderson²,

Barnett³

et al. 2010

Asian J Androl

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Glutamate is a regulated molecule in the mammalian testis. Extracellular regulation of glutamate in the body is determined largely by the expression of plasmalemmal glutamate transporters. We have examined by PCR, western blotting and immunocytochemistry the expression of a panel of sodium-dependent plasmalemmal glutamate transporters in the rat testis. Proteins examined included: glutamate aspartate transporter (GLAST), glutamate transporter 1 (GLT1), excitatory amino acid carrier 1 (EAAC1), excitatory amino acid transporter 4 (EAAT4) and EAAT5. We demonstrate that many of the glutamate transporters in the testis are alternately spliced. GLAST is present as exon-3-and exon-9-skipping forms. GLT1 was similarly present as the alternately spliced forms GLT1b and GLT1c, whereas the abundant brain form (GLT1a) was detectable only at the mRNA level. EAAT5 was also strongly expressed, whereas EAAC1 and EAAT4 were absent. These patterns of expression were compared with the patterns of endogenous glutamate localization and with patterns of D-aspartate accumulation, as assessed by immunocytochemistry. The presence of multiple glutamate transporters in the testis, including unusually spliced forms, suggests that glutamate homeostasis may be critical in this organ. The apparent presence of many of these transporters in the testis and sperm may indicate a need for glutamate transport by such cells.

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Extremely high titre polyclonal antisera against small neurotransmitter molecules: rapid production, characterisation and use in lightand electron-microscopic immunocytochemistry

Cited by 132 publications

References 13 publications

Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons

Molecular Cloning and Functional Identification of Mouse Vesicular Glutamate Transporter 3 and Its Expression in Subsets of Novel Excitatory Neurons

Neuronal nitric oxide synthase alternatively spliced forms: Prominent functional localizations in the brain

Expression of multiple glutamate transporter splice variants in the rodent testis

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