Identification of mitochondrial and non-mitochondrial glutaminase within select neurons and glia of rat forebrain by electron microscopic immunocytochemistry.
Antibodies against the mitochondrial enzyme glutaminase (EC 3.5.1.2), have been used in previous immunocytochemical studies to help identify glutamate-releasing neurons among all glutamate-containing neurons. The studies were based on the idea that glutaminase is enriched within the releasable "transmitter" pools of glutamate. However, evidence is also available to suggest that the enzyme does not occur exclusively within glutamate-releasing neurons. Thus we sought to determine whether glutaminase was immunocytochemically detectable within presynaptic terminals forming asymmetric (putatively excitatory) synapses or, alternatively, occurs in association with mitochondria throughout the cell. For this purpose, we examined the cellular and subcellular distribution of glutaminase- immunoreactivity in neocortical (visual and somatosensory) areas known to contain glutamatergic perikarya. This localization was compared with the distribution in striatal (caudate-putamen and nucleus accumbens) regions recognized to contain high densities of glutamatergic terminals but fewer, if any, glutamatergic perikarya. Glutaminase-immunoreactive perikarya were numerous within the infragranular laminae of neocortex (approximately 1 per 1,000 microns 2 tissue area) but sparse within the caudate-putamen nuclei and accumbens nuclei (less than 1 per 20,000 microns 2.). In addition, heterogeneous distribution of small (less than 1 microns) punctate immunoreactive structures was notable. Relatively high densities of these punctate structures occurred within the supragranular laminae of neocortex, dorsolateral quadrant of the caudate-putamen nuclei, and surrounding certain groups of myelinated fiber bundles throughout the striatum. Electron microscopy revealed diffusely distributed peroxidase immunoreactivity in a select population of dendritic spines, glial processes, and axons. Eight percent of all synapses within the supra-granular laminae were formed by terminals labeled for glutaminase. These principally formed asymmetric junctions on spiny processes. When tissue was incubated with the antibody in the presence of a permeabilizing agent, Photo-flo, high levels of glutaminase immunoreactivity was detectable by electron microscopy within select mitochondria of neocortical (4%) and striatal (8%) perikarya and dendrites, while the diffuse distribution of immunoreactivity within axons and glia was greatly diminished. The differential ultrastructural conditions provide direct demonstration that glutaminase in brain occurs in at least two forms discriminable by their diffuse distribution within non-mitochondrial cytoplasm versus discrete localization within mitochondria. The morphological characteristics of synapses formed by axons exhibiting diffuse distributions of glutaminase immunoreactivity are consistent with the idea that glutaminase-enriched terminals mediate excitatory chemical transmission via the release of glutamate. Because glia containing glutaminase occur juxtaposed to the asymmetric junctions, the glia may utilize neuronally released glutamate for energy metabolism.
Aoki
C
C
Department of Neurology and Neuroscience, Cornell University Medical College, New York, NY 10021.
Kaneko
T
T
Starr
A
A
Pickel
V M
VM
D000818:Animals; D000911:Antibodies, Monoclonal; D002540:Cerebral Cortex / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D003342:Corpus Striatum / Q000276:immunology / Q000378:metabolism / Q000648:ultrastructure; D005972:Glutaminase / Q000032:analysis*; D007124:Immunoenzyme Techniques; D007075:Immunoglobulin M / Q000276:immunology; D007150:Immunohistochemistry; D008297:Male; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000201:enzymology* / Q000648:ultrastructure; D009457:Neuroglia / Q000201:enzymology* / Q000648:ultrastructure; D009474:Neurons / Q000201:enzymology* / Q000648:ultrastructure; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D007075:Immunoglobulin M; D011092:Polyethylene Glycols; D017830:Octoxynol; D005972:Glutaminase