Effects of fractalkine on morphine and/or HIV-1 Tat-induced toxicity was repeatedly tracked in individual striatal neurons (A-E). A within-subjects design and computer-assisted, time-lapse tracking of individual neurons is used to compare the survival of the same neuron before and at 20 min intervals throughout treatments (A-E). Thriving neurons are evident in control (A; neurons 1-3) or morphine (Morph) + Tat-treated cultures co-administered fractalkine (B; neurons 1-4) at 48 h. By contrast, increased neurodegeneration and death was apparent with morphine plus Tat exposure (C). Neuron death is often preceded by the systematic degeneration of neurites and fragmentation of the cell body (Tat + morphine treatment; arrowheads, neuron 1), while death per se occurs precipitously; denoted here by fragmentation of the cell body and some loss of birefringence (arrow, neuron 1; 44 h and 48 h) (C). Some individual microglia that were tracked are encircled in red, orange, and yellow, respectively (arrowheads indicate prior movement); there was close association of microglia with the dying (1) and surviving (2) neuron (not all microglia in the fields are encircled) (C). Neuron death has been confirmed using other viability markers (see text). Many neurons remain viable despite morphine and Tat co-exposure (e.g., neuron 2); some surrounding cells, including astroglia, immature glial precursors and microglia, can display sporadic movement; scale bars = 20 μm. While Tat alone did not cause significant neurotoxicity relative to controls by 48 h [61, 62]; there was a significant interaction when overall neuron losses were compared in Tat versus non-Tat vehicle-treated cultures (§P < 0.03; Tat × time vs. Vehicle-control × time). Compared to controls or exposure to fractalkine alone, combined morphine and Tat (Morph+Tat) treatment increased neuronal death at 48 h (D) (*P < 0.05 vs. controls). Exogenous fractalkine (1 μg/ml) (red markers and connecting lines) prevented the accelerated neuronal death caused by combined Morph+Tat (#P < 0.05 vs. Morph+Tat) (D). By contrast, CX3CR1 immunoblockade (blue markers and connecting lines) by itself caused significant neurotoxicity that was indistinguishable from the combined effects of Morph+Tat (E) (*P < 0.05 vs. controls). Moreover, when CX3CR1 blockade was combined with Morph+Tat, there were no additive neuron losses (E). The findings suggest a critical role for fractalkine-CX3CR1 signalling in potentiating the toxic effects of opioids in Tat-exposed neurons; data are the mean number of surviving neurons (compared to pretreatment numbers) ± SEM from n = 3-6 experiments; wild-type (WT) neurons and mixed glia; antibodies (Ab); scale bar = 20 μm.