Dynamic cytoskeletal rearrangements are involved in neuronal growth cone motility and guidance. To investigate how cell surface receptors translate guidance cue recognition into these cytoskeletal changes, we developed a novel in vitro assay where beads, coated with antibodies to the immunoglobulin superfamily cell adhesion molecule apCAM or with purified native apCAM, replaced cellular substrates. These beads associated with retrograde F-actin flow, but in contrast to previous studies, were then physically restrained with a microneedle to simulate interactions with noncompliant cellular substrates. After a latency period of ∼10 min, we observed an abrupt increase in bead-restraining tension accompanied by direct extension of the microtubule-rich central domain toward sites of apCAM bead binding. Most importantly, we found that retrograde F-actin flow was attenuated only after restraining tension had increased and only in the bead interaction axis where preferential microtubule extension occurred. These cytoskeletal and structural changes are very similar to those reported for growth cone interactions with physiological targets. Immunolocalization using an antibody against the cytoplasmic domain of apCAM revealed accumulation of the transmembrane isoform of apCAM around bead-binding sites. Our results provide direct evidence for a mechanical continuum from apCAM bead substrates through the peripheral domain to the central cytoplasmic domain. By modulating functional linkage to the underlying actin cytoskeleton, cell surface receptors such as apCAM appear to enable the application of tensioning forces to extracellular substrates, providing a mechanism for transducing retrograde flow into guided growth cone movement.

Summary:Purpose: Several findings suggest that energy metabolism and the glutamate-glutamine cycle may be impaired in epilepsy. Positron emission tomography often shows interictal hypometabolism of the epileptogenic hippocampus. In vivo microdialysis studies show that seizure-associated glutamate release is doubled, and clearance is slowed. We hypothesized that the glutamate-glutamine cycle between neurons and glia may be decreased in the epileptic human hippocampus.Methods: A 20% solution of 2-13 C-glucose was infused before resection of the epileptogenic hippocampus. Blood glucose isotopic fractions were measured every 30 min. Blood and brain specimens were frozen quickly; perchloric acid extracts of the small metabolites were prepared and analyzed by proton and carbon magnetic resonance spectroscopy (MRS) at 11.75 Tesla.Results: Standard histology showed 12 with hippocampal sclerosis and five with minimal neuron loss. The relative rates of glutamate-glutamine cycling with respect to glutamate synthesis were decreased in biopsies affected by hippocampal sclerosis (mean, 0.08; 95% confidence interval, 0.04-0.12) compared with those with minimal neuron loss (0.52; 95% CI, 0.30-0.75). Mean cellular glutamate concentrations were higher in minimal neuron loss (8.9 mM; 95% CI, 7.4-10.4) than hippocampal sclerosis (7.3 mM; 95% CI, 5.9-8.7). Cellular glutamine concentrations (mean, 2.8 mM; 95% CI, 2.4-3.2; n ‫ס‬ 17) were the same in all groups.Conclusions: The epileptogenic, gliotic human hippocampus appears to be characterized metabolically by slow rates of glutamate-glutamine cycling, decreased glutamine content, and a relative increase in glutamate content. We hypothesize that the low rate of glutamate-glutamine cycling that results from a failure of glial glutamate detoxification could account for slow glutamate clearance from synapses and continuing low-grade excitotoxicity.

Mesial temporal lobe epilepsy is characterized by hippocampal atrophy, hypometabolism, and decreased N-acetylaspartate, often attributed to neuron loss and gliosis. Twenty hippocampal specimens were obtained during temporal lobectomy and frozen quickly. Perchloric acid extracts of the small metabolites were analyzed by proton magnetic resonance spectroscopy. There were no significant associations between hippocampal neuron loss and the cellular content of N-acetylaspartate, glutamate, GABA, glutamine, or aspartate. The mean metabolite content of hippocampi with less than 30% of neurons remaining was the same as those with greater than 65% of neurons surviving. Mean N-acetylaspartate levels were below those reported by in vivo studies of control subjects. The highest and the lowest glutamate concentrations were seen in specimens with the worst neuron loss. A highly significant association between hippocampal N-acetylaspartate and glutamate content was seen with weak associations between N-acetylaspartate and aspartate and glutamate and aspartate. The hippocampal content of N-acetylaspartate, glutamate, GABA, glutamine, and aspartate is altered minimally by severe neuron loss in mesial temporal lobe epilepsy. The epileptic human hippocampus has increased intracellular glutamate content that may contribute to the epileptogenic nature of hippocampal sclerosis.

Plectin is a high molecular weight protein originally identified and characterized as a major cytoskeletal component of the C6 rat glioma cell. Here we demonstrate by immunoblotting of crude intermediate filament (IF) protein preparations that plectin is a cytoskeleton-associated component of the rat spinal cord. We then used avidin-biotin peroxidase immunocytochemistry and indirect immunofluorescence to localize plectin within the adult rat central nervous system (CNS) and examine its distribution with respect to IF proteins. Plectin immunoreactivity is localized to all ependymal cells including the choroidal epithelial cells and tanycytes, Bergmann glial processes, radially oriented glial cells in the spinal cord, astrocytes in white matter, a subset of astrocytes in gray matter, a subset of motoneurons in the brainstem and spinal cord, and certain endothelial cells. Colocalization studies with neural IF proteins show that plectin has a unique distribution pattern which most closely resembles, but is distinct from, that of vimentin. The few plectin positive neurons invariably also contain the neurofilament triplet proteins and peripherin, so that the ability of plectin to bind to the triplet proteins in vitro may reflect an in vivo interaction. The predominance of plectin at the inner ventricular boundaries of the nervous system as well as at the blood-brain barrier is in line with the pattern of plectin expression in other tissues and suggests a general role for plectin in the maintenance of such junctional regions.