Neuron-specific enolase (NSE) and β-tubulin III (also referred to as TuJ1) immunoreactivity in hippocampal cultures. Cells were incubated with chicken antibodies against either NSE or β-tubulin III (Left). Neurons show strong immunoreactivity, as expected from neuron-specific markers, while astrocytes surrounding neurons display faint fluorescence emission. Cells submitted to the immunocytochemistry procedure, but with the omission of the primary antibodies, serve as control for background fluorescence (Right). Scale bar, 10μm.

Water soluble graft co-polymer SWNT-PEG does not affect the endocytotic load of the recycling dye FM1−43 during constitutive recycling. A) The application of FM1−43 labels membranes (load), internalized membrane remains labeled after washout of the dye (rinse). In the loading phase, the dye is taken up by endocytosis. B) Time course of the experiment outlined in (A). The arrow indicates the beginning of the 5 minute FM1−43 application (broken abscissa). The peak fluorescence signal is an indication of the extent of FM1−43 endocytotic load. The signal decays during rinsing phase reaching a steady-state level, which reports on the fluorescence of FM1−43 loaded in vesicular compartments. Treatment of neurons with different concentrations of SWNT-PEG, for 3 days in culture and throughout the entire course of experiment, did not affect the endocytotic load of FM1−43 (grey/black squares) when compared to untreated neurons (control; open squares). Images show DIC of a neuron, its fluorescence before (0s), immediately after addition of FM1−43 (400s) and at steady-state (900s). Numbers in parenthesis indicate the number of neurons studied in each condition. Points and bars represent means ± standard error of the mean. Concentrations of SWNT-PEG are given in μg/mL. Scale bar, 10μm.

SWNT-PEG reduces endocytotic load of the recycling dye FM1−43 in stimulated neurons. A) Time course of the experiment using HiK⁺ solution to stimulate exocytosis. The arrow indicates the beginning of the 5 minute FM1−43/HiK⁺ application (broken abscissa). Treatment of neurons with SWNT-PEG, for 3 days in culture and throughout the entire course of experiment, reduced the stimulated endocytotic load of FM1−43 (black squares) when compared to untreated neurons (control; open squares). B) This effect of SWNT-PEG on the endocytotic load (plasma membrane and vesicular compartment; peak response) is dose-dependent (grey bars) and is not seen in unstimulated cells (black bars). C) The steady-state level of FM 1−43 dye retention reports on vesicular compartments. SWNT-PEG treatment reduced steady-state dye retention in stimulated (grey bars), but not in unstimulated (black bars) neurons. Points and bars represent means ± standard error of the mean. Concentrations of SWNT-PEG are given in μg/mL. Numbers in parenthesis indicate the number of neurons studied in each condition; the number of cells in C are the same as in B. Asterisks indicate a significant difference in measurement when compared to control (one-way ANOVA followed by Fisher's LSD test; *p<0.05, **p<0.01).

Soluble SWNTs preferably affect endocytosis over exocytosis. (Left) Peak FM1−43 fluorescence indicates the extent of plasma membrane and vesicular load. Using HiK⁺ solution to stimulate neurons in the presence of FM1−43 (FM1−43/HiK⁺) shows a large difference in the cellular load between control (open squares) and SWNT-PEG treated cells (black squares). The second application of HiK⁺ solution at the end of the experiment stimulates exocytosis of FM1−43 loaded in vesicles. The arrows (from left to right) indicate the beginning of the 5 minute FM1−43/HiK⁺ application (broken abscissa) and the second application of HiK⁺ alone lasting 200 s (horizontal line). (Right) The rate of FM1−43 de-staining during second application of HiK⁺ is faster and significantly more pronounced in SWNT-PEG treated neurons when compared to their controls. Note that FM1−43 fluorescence emission was normalized to the steady-state level just prior to the application of HiK⁺. Points and bars represent means ± standard error of the mean. Asterisks indicate a significant difference in measurement when compared to time-matched control (Student t-test; **p<0.01).

Possible mechanism of soluble SWNTs action on endocytosis. An increase in total membrane area during neurite outgrowth occurs by incorporating new membrane by vesicle fusion or exocytosis. Membrane can also be retrieved by the reverse process of endocytosis. When neurons are exposed to soluble carbon nanotubes endocytosis is impaired effectively allowing for the net insertion of plasma membrane.