Methamphetamine intoxication causes long-lasting damage to dopamine nerve endings in the striatum. The mechanisms underlying this neurotoxicity are not known but oxidative stress has been implicated. Microglia are the major antigen-presenting cells in brain and when activated, they secrete an array of factors that cause neuronal damage. Surprisingly, very little work has been directed at the study of microglial activation as part of the methamphetamine neurotoxic cascade. We report here that methamphetamine activates microglia in a dose-related manner and along a time course that is coincident with dopamine nerve ending damage. Prevention of methamphetamine toxicity by maintaining treated mice at low ambient temperature prevents drug-induced microglial activation. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), which damages dopamine nerve endings and cell bodies, causes extensive microglial activation in striatum as well as in the substantia nigra. In contrast, methamphetamine causes neither microglial activation in the substantia nigra nor dopamine cell body damage. Dopamine transporter antagonists (cocaine, WIN 35,428 [(Ϫ)-2-␤-carbomethoxy-3-␤-(4-fluorophenyl)tropane 1,5-naphthalenedisulfonate], and nomifensine), selective D1 (SKF 82958 [(Ϯ)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide]), D2 (quinpirole), or mixed D1/D2 receptor agonists (apomorphine) do not mimic the effect of methamphetamine on microglia. Hyperthermia, a prominent and dangerous clinical response to methamphetamine intoxication, was also ruled out as the cause of microglial activation. Together, these data suggest that microglial activation represents an early step in methamphetamine-induced neurotoxicity. Other neurochemical effects resulting from methamphetamine-induced overflow of DA into the synapse, but which are not neurotoxic, do not play a role in this response.

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Membrane sheets elaborated by cultured murine oligodendroglia provide a unique system for examining associations between myelin proteins and cytoskeletal elements. Interactions can be observed and manipulated more readily than in the multilamellar myelin membrane in vivo. Immunocytochemical staining of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) shows that it is distributed diffusely in some regions of membrane sheets, but colocalized with tubulin in lacy networks and major veins in other regions. Staining with phalloidin also reveals two distributions of F-actin: 1) small aggregates within the diffuse CNPase regions and 2) filaments colocalized with tubulin and CNPase in the lacy networks and veins. Application of colchicine at 10 micrograms/ml for 4 hr disrupts microtubular structures in the lacy network, while those in major veins remain intact. This suggests that microtubules in the lacy network are treadmilling more rapidly than those in the major veins. The distribution of CNPase and F-actin is not altered under these conditions. In contrast, cytochalasin B disrupts F-actin, microtubules, and CNPase in the lacy networks, indicating that cross-linking between these three proteins is disrupted. Both colchicine and cytochalasin B cause fusion of myelin basic protein (MBP) domains in membrane sheets. This appears to be a consequence of disruption of microtubules in the lacy networks, which normally outline the MBP domains. In summary, these results provide evidence for 1) direct association of CNPase with F-actin and tubulin in cytoskeletal structures and 2) organization of MBP into domains via association with microtubules in the lacy networks.

Antibodies to galactocerebroside (GalC) cause major changes in the organization of the membrane sheets elaborated by murine oligodendroglia in culture. Exposure of oligodendroglia to rabbit anti-GalC IgG for 15 min followed by fluoresceinated second antibodies results in patches of surface GalC staining; when second antibodies are applied after 2 hr of anti-GalC, the pattern of staining on membrane sheets is solid and wrinkled. Anti-GalC exposure for 24 hr results in contracted membrane sheets. No membrane contraction is detected in cultures treated with anti-sulfatide IgM or anti-proteolipid protein IgG. In cultures exposed to anti-GalC continuously for 4-7 d, there is a marked decrease in numbers of extended membrane sheets with an accompanying increase in contracted sheets. This effect is reversible upon removal of anti-GalC from the culture media. By scanning electron microscopy, normally flat membrane sheets appear ruffled after 2 hr of anti-GalC treatment; by 24 hr, contracted membrane sheets consist entirely of bulbous protrusions. Oligodendrocyte membranes exposed to anti-sulfatide for 24 hr are not contracted but are covered with bulbous protrusions. The organization of underlying membrane structures was examined in relation to membrane patching and sheet contraction. In membranes with patching induced by exposure to anti-GalC for 15 min, the anti-GalC: GalC complexes are localized over cytoplasmic MBP domains, with the unstained areas located above cytoplasmic microtubular structures. Membrane sheets that are contracted in response to anti-GalC exposure for 6-24 hr show intense GalC staining over microtubular structures. Anti-GalC exposure does not change metabolism of GalC; in cultures incubated with 3H-galactose and anti-GalC for 24 hr, there are no alterations in GalC labeling compared with control cultures. In summary, these results provide direct evidence that interaction between surface glycolipids and external antibodies can initiate a sequence of events leading to dramatic changes within the oligodendrocyte.

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