g-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. This molecule activates both ionotropic GABA_A receptors and metabotropic GABA_B receptors to induce neuronal signaling. Brain GABA levels are closely regulated by GABA degradation enzymes like succinic semialdehyde dehydrogenase (SSADH), preventing excessive rises in GABA concentrations. Indeed, in human SSADH deficiency, both GABA and the metabolite GHB increase in the extracellular space. Since GABA itself is an agonist of both GABA receptors, while GHB is a selective GABA_B agonist, 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 in our cells. Accordingly, the selective GABA_A agonist THIP (10 mM) induced a Ca²⁺ rise of 2.88 ±0.34 DF/F₀ Fluo-4 units (n=10). Co-application of the selective GABA_B agonist baclofen (10 mM) reduced the THIP response to 64 ±9% (n=10, p=0.039). The mixed agonist GABA (10 mM) induced a calcium rise of 2.62 ±0.17 DF/F₀ (n=10) while co-application of the selective GABA_B antagonist CGP55845A (1 mM) increased the GABA response to 3.44 ±0.27 DF/F₀ (n=11, p<0.001). Mimicking the pathophysiological condition seen in SSADH deficiency, GABA (1 mM) caused Ca²⁺ rises of 2.32 ±0.2 DF/F₀ (n=18) that could be reduced by high concentrations (1 mM) of GHB (by 18%±8%, n=15, p=0.038). We conclude that GHB partially protect against GABA induced Ca²⁺ rises in developing cortical neurons, which is important for understanding the pathophysiology of SSADH deficiency.