Pre-embedding Staining for GAD₆₇ Versus Postembedding Staining for GABA as Markers for Central GABAergic Terminals

Abstract

SUMMARY Pre-embedding immunocytochemistry for the active form of glutamate decarboxylase (GAD 67 ) and postembedding staining for ␥-aminobutyric acid (GABA) were compared as markers for central GABAergic terminals in the phrenic motor nucleus, in which phrenic motor neurons had been retrogradely labeled with cholera toxin B-horseradish peroxidase. Nerve terminals with or without GAD 67 immunoreactivity were identified in one ultrathin section. GABA was localized with immunogold in an adjacent section after etching and bleaching. GABA labeling density was assessed over 519 GAD 67 -positive and GAD 67 -negative nerve terminals in the phrenic motor nucleus. Frequency histograms showed that statistically higher densities of gold particles occurred over most GAD 67 -positive terminals. However, some GAD 67 -negative terminals also showed high densities of gold particles, and some GAD 67 -positive terminals showed low densities. Preabsorption of the anti-GABA antibody with a GABA-protein conjugate, but not with other amino acid-protein conjugates, significantly reduced gold labeling over both GAD 67 -positive and GAD 67 -negative terminals. These results show that the presence of GAD 67 immunoreactivity correlates strongly with high densities of immunogold labeling for GABA in nerve terminals in the phrenic motor nucleus. Preabsorption controls indicate that authentic GABA was localized in the postembedding labeling procedure. Only a small proportion of intensely GABAimmunoreactive terminals lack GAD 67 , suggesting that both GAD 67 and GABA are reliable markers of GABAergic nerve terminals. (J Histochem Cytochem 46:1261-1268, 1998) GABAergic nerve terminals throughout the central nervous system have been identified using either preor postembedding immunogold methods to localize ␥-aminobutyric acid (GABA), or pre-embedding immunocytochemistry to visualize glutamate decarboxylase (GAD), the enzyme required for synthesis of GABA from l-glutamate (McLaughlin et al. 1975;Gabbott et al. 1986;Babb et al. 1988;Zhan et al. 1989; Helfert et al. 1992;Tai and Goshgarian 1996). A general presumption is that all nerve terminals containing GAD 67 , the active form of the enzyme, contain high levels of GABA, but this relationship has not been analyzed quantitatively. Maxwell et al. (1989) showed that, in the lateral cervical nucleus of the cat, GAD-immunoreactive terminals exhibited high densities of gold labeling for GABA but did not show quantitatively whether the density of GABA labeling was high over all GAD-positive terminals and low over all GAD-negative terminals. We have previously used tissue fixed with 2.5% glutaraldehyde to localize GAD 67 immunoreactivity and showed that it was suitable for immunogold localization of the amino acid neurotransmitter glutamate (Murphy et al. 1996).Using similarly processed tissue in this study, we determined whether GAD 67 -containing nerve terminals labeled by pre-embedding immunocytochemistry showed high densities of gold particles after postembedding im-

Sphingosine-1-Phosphate-Induced Nociceptor Excitation and Ongoing Pain Behavior in Mice and Humans Is Largely Mediated by S1P3 Receptor

“…The sphingolipid S1P is a multifaceted immune modulator that can act extracellularly in an autocrine or paracrine manner or intracellularly as a second messenger molecule (Mitra et al, 2006;Alvarez et al, 2007;Takabe et al, 2008). Extracellular and intracellular levels of S1P are tightly regulated by sphingosine kinases (SphKs) and S1P degrading enzymes van Echten-Deckert and Herget, 2006).…”

“…S1P is deactivated by S1P-phosphatases or degraded by S1P-lyase (Pyne and Pyne, 2000;Le Stunff et al, 2004;Bandhuvula and Saba, 2007). Mice deficient in both SphK 1 and SphK 2 are not viable, indicating an essential cellular requirement for S1P (Mizugishi et al, 2005).…”

“…Chemoattractants, such as TNF␣ or NGF, can stimulate S1P production in immune cells, epithelia, and even neurons (Zhang et al, 2006b;Alemany et al, 2007), which can release S1P presumably via the specific multidrug resistanceassociated protein ABCC1 (Mitra et al, 2006). Although S1P in blood plasma may reach up to micromolar concentrations, it is largely bound to plasma proteins, and free active S1P concentrations are probably very low (Murata et al, 2000;Schmidt et al, 2006;Ohkawa et al, 2008).…”

“…Although S1P in blood plasma may reach up to micromolar concentrations, it is largely bound to plasma proteins, and free active S1P concentrations are probably very low (Murata et al, 2000;Schmidt et al, 2006;Ohkawa et al, 2008). However, high concentrations of free S1P can, however, arise locally at inflammation sites and presumably at sites of thrombocyte activation (Mitra et al, 2006;Hammad et al, 2008;Ulrych et al, 2011).…”

“…Human platelets contain high concentrations of sphingomyelin-derived sphingosine-1-phosphate (S1P), which can be released upon activation and shape change (Dahm et al, 2006;Ulrych et al, 2011). In blood plasma, S1P levels may reach up to micromolar concentrations, most of which is bound to plasma proteins (Murata et al, 2000;Schmidt et al, 2006;Ohkawa et al, 2008). S1P acts as a lipid growth factor and induces robust endothelial cell activation, resulting in cellular locomotion, vascular maturation, and angiogenesis (Daum et al, 2009).…”