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Mycobacterium tuberculosis (Mtb) encounters stresses during the pathogenesis and treatment of tuberculosis (TB) that can suppress replication of the bacteria and render them phenotypically tolerant to most available drugs. Where studied, the majority of Mtb in the sputum of most untreated subjects with active TB have been found to be nonreplicating by the criterion that they do not grow as colony-forming units (cfus) when plated on agar. However, these cells are viable because they grow when diluted in liquid media. A method for generating such "differentially detectable" (DD) Mtb in vitro would aid studies of the biology and drug susceptibility of this population, but lack of independent confirmation of reported methods has contributed to skepticism about their existence. Here, we identified confounding artifacts that, when avoided, allowed development of a reliable method of producing cultures of ≥90% DD Mtb in starved cells. We then characterized several drugs according to whether they contribute to the generation of DD Mtb or kill them. Of the agents tested, rifamycins led to DD Mtb generation, an effect lacking in a rifampin-resistant strain with a mutation in rpoB, which encodes the canonical rifampin target, the β subunit of RNA polymerase. In contrast, thioridazine did not generate DD Mtb from starved cells but killed those generated by rifampin.Mycobacterium tuberculosis | differentially detectable | phenotypic tolerance | rifampin | thioridazine

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The GABA synthesizing enzyme, glutamate decarboxylase (GAD), has been localized by light and electron microscopy in the rat lumbosacral spinal cord using a peroxidase-labeling antibody technique. The light microscopic localization shows heavy, punctate reaction product for GAD in the dorsal horn laminae I-III. Moderately heavy reaction product is also seen in the deeper dorsal horn laminae IV-VI, the medial aspect of the intermediate gray (lamina VII) and the region around the central canal (lamina X). A moderately light concentration of GAD reaction product is observed in the ventral horn, and punctate deposits of reaction product also are seen on motoneuron cell bodies. The punctate distribution of reaction product for GAD in both ventral and dorsal horns, as visualized by light microscopy, corresponds to GAD-containing synaptic terminals seen by electron microscopy in comparable regions of the spinal gray. Many more GAD-positive terminals are observed in dorsal horn laminae I-III than in deeper laminae IV-VI. GAD-containing terminals in the dorsal horn are presynpatic to dendrites and cell bodies. Gad-containing terminals presynaptic to other axon terminals are observed also, and they are more numerous in laminae II and III. In the ventral horn motor nuclei, GAD-positive knobs are presynaptic to large and small dendrites and motoneuror cell bodies. In addition, small GAD-containing terminals also are presynaptic to larger axonal terminals which are in turn presynaptic to motoneuron somata. The observation of GAD-containing terminals presynaptic to dendrites and cell bodies in both dorsal and ventral horns is compatible with the evidence suggesting that GABA terminals may mediate postsynaptic inhibition of spinal interneurons and motoneurons. The additional finding of GAD-positive terminals presynaptic to other axonal terminals in the dorsal horn and motor nuclei is consistent with the growing evidence that GABA also may be the transmises mediating presynaptic inhibition via axo-axond synapses in the spinal cord.

Glutamate decarboxylase (GAD), the enzyme that synthesizes the neurotransmitter gamma-aminobutyric acid (GABA), has been localized in the rat olfactory bulb by immunocytochemical methods with both light and electron microscopy. The light microscopic results demonstrated GAD-positive puncta concentrated in the external plexiform layer and in the glomeruli of the glomerular layer. In addition, GAD-positive reaction product stained the dentrites and somata of granule and periglomerular cells. The electron microscopic observations confirmed the presence of GAD-positive reaction product within granule and periglomerular somata and dendrites. In electron micrographs of the external plexiform layer, the gemmules which arise from the distal dentrites of granule cells were also observed to be filled with reaction product, and these structures corresponded in size and location to the puncta observed in light microscopic preparations. The gemmules were observed to form reciprocal dendrodentritic synaptic junctions with mitral cell dentrites which lacked reaction product. In the glomeruli, GAD-positive reaction product was observed in the dentritic shafts and gemmules of periglomerular cells which also formed reciprocal dendrodentritic synaptic contacts with mitral/tufted cell dentrites. The localization of GAD in known inhibitory neurons of the olfactory bulb supports the case that these local circuit neurons use GABA as their neurotransmitter. The present study demonstrates that GAD molecules located within certain neuronal somata and dentrites can be visualized with antisera prepared against GAD that was purified from synaptosomal fractions of mouse brains. This finding suggests that the lack of GAD staining within somata and dentrites of GABA-ergic neurons noted in previous studies of the cerebellum and spinal cord was probably due to low GAD concentrations, rather than to antigenic differences among GAD molecules located in different portions of the neuron. A striking differences among GAD molecules located in different portions of the neuron. A striking difference between the granule and periglomerular neurons of the olfactory bulb and the neurons of the cerebellum and spinal cord is that the former have presynaptic dentrites while the latter do not. Since GAD-positive reaction product can be detected in the somata and dentrites of GABA-ergic neurons which have presynaptic dentrites, it is suggested that these neurons may differ from other GABA-ergic neurons with respect to either transport or metabolism of GAD.