Glutamate is partially responsible for HIV/MDM neurotoxicity. Supernatants were filtered through membranes with molecular weight cutoffs of 50 kDa (<50 kDa), 10 kDa (<10 kDa), and 3 kDa (<3 kDa) and assayed for neurotoxicity on DIV 21 hippocampal cultures (A). The neurotoxicity of factors larger than 3 kDa in the supernatants (>3 kDa) was determined by resuspending the filter retentate in macrophage media in the same volume as the filtrate. B, Supernatants were exposed to proteinase-K (1 μg/ml for 2 h at 37°C), and then treated supernatants were filtered through a 3 kDa filter before testing for neurotoxicity on DIV 21 hippocampal cultures. C, Supernatants were boiled (10 min; ΔH), cooled to 37°C, and tested for neurotoxicity on DIV 21 hippocampal cultures. Neither proteinase-K treatment nor boiling abrogated neurotoxicity (*p < 0.01 vs mock; **p < 0.01 vs mock treated). D, The contribution of glutamate from HIV-Jago supernatants was assayed by enzymatic degradation of glutamate in supernatants by treatment with GPT. Control media was spiked with 100 μm glutamate alone. Control and MDM supernatants (mock and HIV-Jago) were treated with GPT, added to DIV 21 hippocampal cultures (1:10 dilution for 24 h for a final dilution of 10 μm glutamate), and followed by MAP-2 staining and cell counting (^#p < 0.01 vs GPT treated).