Cellular and molecular responses to acute cocaine treatment in neuronal-like N2a cells: potential mechanism for its resistance in cell death

Abstract

Cocaine is a highly abused drug that causes psychiatric and neurological problems. Its entry into neurons could alter cell-biochemistry and contribute in the manifestation of early pathological symptoms. We have previously shown the acute cocaine effects in rat C6 astroglia-like cells and found that these cells were highly sensitive to cocaine in terms of manifesting certain pathologies known to underlie psychological disorders. The present study was aimed to discern acute cocaine effects on the early onset of various changes in Neuro-2a (N2a) cells. Whole-cell patch-clamp recording of differentiated cells displayed the functional voltage-gated Na+ and K+ channels, which demonstrated the neuronal characteristics of the cells. Treatment of these cells with acute cocaine (1 h) at in vivo (nM to μM) and in vitro (mM) concentrations revealed that the cells remained almost 100% viable. Cocaine administration at 6.25 μM or 4 mM doses significantly reduced the inward currents but had no significant effect on outward currents, indicating the Na+ channel-blocking activity of cocaine. While no morphological change was observed at in vivo doses, treatment at in vitro doses altered the morphology, damaged the neurites, and induced cytoplasmic vacuoles; furthermore, general mitochondrial activity and membrane potential were significantly decreased. Mitochondrial dysfunction enabled the cells switch to anaerobic glycolysis, evidenced by dose-dependent increases in lactate and H2S, resulting unaltered ATP level in the cells. Further investigation on the mechanism of action unfolded that the cell’s resistance to cocaine was through the activation of nuclear factor E2-related factor-2 (Nrf-2) gene and subsequent increase of antioxidants (glutathione [GSH], catalase and GSH peroxidase [GPx]). The data clearly indicate that the cells employed a detoxifying strategy against cocaine. On a broader perspective, we envision that extrapolating the knowledge of neuronal resistance to central nervous system (CNS) diseases could delay their onset or progression.

Cocaine Induces Cytoskeletal Changes in Cardiac Myocytes: Implications for Cardiac Morphology

“…The cocaine concentration chosen by this study was 1 µg/mL, compared to previous literature relating to both cardiac and non-cardiac in vitro models this dosage lies within the plasma ranges conventionally observed in recreational drug users [ 12 , 19 , 20 , 21 , 22 , 23 , 24 ]. The cocaine treatment selected in the present study was also considered in relation to reports that have shown insufflation of 1.5 mg/kg (equating to 105 mg/70 kg person) results in peak urine concentrations of 0.3 µg/mL with post-mortem results highlighting mortality in 100 cases at cocaine concentrations of 9.8 1 µg/mL) and other studies demonstrating cocaine induced apoptosis between 0.1–1.0 µmol/mL treatments [ 25 , 26 , 27 ].…”

Cocaine potentiates an inflammatory response in C6 astroglia‑like cells

“…Initially the effect of treatment with cocaine at concentrations ranging from 0.5 to 7 mM for 24 and 48 h on cell viability was assessed. The selection of cocaine concentrations was based on earlier reports ( 20 , 23 ), and the lag period and doubling time of these cells ( 17 ) were used as the criteria for treatment durations of 24 and 48 h. Compared with the corresponding controls, cocaine significantly decreased the cell viability at these time points ( Fig. 1A and B ; P<0.01 or P<0.001).…”

Ethanol and cocaine increases microtubule stability and decreases [11C]MPC-6827 uptake in SH-SY5Y cells

“…To investigate the effects of cocaine and alcohol on tubulin dynamics, we performed a MT-based assay (Cytoskeleton, Inc., Denver, CO, USA) [25][26][27] in SH-SY5Y neuroblastoma cells treated with 100 mM EtOH [10] and 1 mM cocaine [8] (n=6/group) for 3 days. This commercially available kit separates large complexes of polymerized MTs attached to nuclei and Golgi bodies into bound and non-polymerized free tubulins.…”

“…We performed cell binding assays in vitro in SH-SY5Y cells with [ 11 C]MPC-6827 following our previously published protocols. [28][29][30] The cells were treated with 100 mM EtOH or 1 mM cocaine [8] (n = 6/group) for 3 days. We then measured radiotracer binding by adding [ 11 C]MPC-6827 (1-2 µCi/well) and incubating the cells for 5, 30, 60, and 90 min at room temperature (n = 6/time point).…”

“…For example, repeated cocaine administration has been shown to change stathmin, a regulatory protein crucial to MT dynamics, [7,6] causing morphologic changes. [8] Neuronal structural changes may contribute to the progression of AUD and SUDs. [9] Chronic ethanol (EtOH) exposure signi cantly stabilizes neuronal and acetylated MTs in hepatic PC12 cells, [10] increases dendrite lengths and neurite outgrowth and causes aberrant sprouting of hippocampal neurites.…”