Background <p>New psychoactive substances (NPSs) have been implicated in abuse and crime, but their effects on the central nervous system remain poorly understood.</p> Objective <p>To evaluate the impact of four NPSs (3,4-dichloromethylphenidate (DCMP), 4-EA-NBOMe, LY-2183240, and AB-CHFUPYCA) on neural function using multiple experimental approaches alongside reference drugs (methamphetamine, cocaine, and tetrahydrocannabinol).</p> Results <p>Morphological analysis revealed that LY-2183240 and AB-CHFUPYCA specifically induced abnormal astrocyte morphology, while DCMP and 4-EA-NBOMe reduced VGLUT1-positive puncta numbers. Using synaptic channel sensors, 4-EA-NBOMe showed responses in both glutamate (GluR1L497Y) and dopamine (GRAB_DA2m) sensors, while LY-2183240 activated only the dopamine sensor. None of the NPSs activated GABAc receptors.</p> Conclusion <p>NPSs exhibit diverse effects on neural cells and synaptic function, demonstrating cell-type-specific responses and selective activation of synaptic channels. These findings provide insights into NPS effects on the central nervous system and their potential mechanisms of toxicity.</p>