High-energy, short-duration electric pulses (EPs) are known to be effective in neuromodulation, but the biological mechanisms underlying this effect remain unclear. Recently, we discovered that nanosecond electric pulses (nsEPs) could initiate the phosphatidylinositol_4,5-bisphosphate (PIP₂) depletion in non-excitable cells identical to agonist-induced activation of the G_q11 coupled receptors. PIP₂ is the precursor for multiple intracellular second messengers critically involved in the regulation of intracellular Ca²⁺ homeostasis and plasma membrane (PM) ion channels responsible for the control of neuronal excitability. In this paper we demonstrate a novel finding that five day in vitro (DIV5) primary hippocampal neurons (PHNs) undergo significantly higher PIP₂ depletion after 7.5 kV/cm 600 ns EP exposure than DIV1 PHNs and day 1–5 (D1-D5) non-excitable Chinese hamster ovarian cells with muscarinic receptor 1 (CHO-hM₁). Despite the age of development, the stronger 15 kV/cm 600 ns or longer 7.5 kV/cm 12 µs EP initiated profound PIP₂ depletion in all cells studied, outlining damage of the cellular PM and electroporation. Therefore, the intrinsic properties of PHNs in concert with nanoporation explain the stronger neuronal response to nsEP at lower intensity exposures. PIP₂ reduction in neurons could be a primary biological mechanism responsible for the stimulation or inhibition of neuronal tissues.