Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. GABA activates both ionotropic GABA_A receptors and metabotropic GABA_B receptors to induce neuronal signaling. The levels of GABA in the brain are closely regulated by enzymes such as succinic semialdehyde dehydrogenase (SSADH), preventing excessive rises in GABA concentrations. Indeed, in human SSADH deficiency, GABA and its catabolite GHB increases in the extracellular space. Since GHB is a selective GABA_B agonist, where GABA activates both GABA_A and GABA_B receptors, SSADH deficiency represents a unique pathophysiological condition. However, the combined effect of excess GABA and GHB on neuronal signaling is poorly understood. Using Ca²⁺ imaging in cultured cortical mouse neurons (4-10 days in vitro), we first confirmed that both GABA_A and GABA_B receptors were functionally expressed on the cultured cells by exposing them to GABA or the selective GABA_A and GABA_B agonists THIP and baclofen. Next, we mimicked the pathophysiological condition seen in SSADH deficiency, and found that GABA caused Ca²⁺ rises in young neurons that could be reduced by moderate to high concentrations of GHB (100-1000 mM). Furthermore, GABA and GHB were able to induce calcium transients in astrocytes. All actions of GHB could be blocked by the GABA_B antagonist CGP55845, making it unlikely that GHB affects Ca²⁺ levels via a distinct, high-affinity GHB binding site. In conclusion, we have revealed that GHB partially protects against GABA induced Ca²⁺ rises in developing cortical mouse neurons. At the same time GHB induces Ca²⁺ rises in cortical astrocytes, which might be relevant under certain physiological or pathophysiological condition during brain development. Our findings also have relevance for understanding interactions between GABA and GHB in SSADH deficiency.