<p>The interaction between neurons and glial cells is garnering increased focus due to its significance in understanding neurodegenerative disorders. Calcium ion (Ca²⁺)-signaling through G-protein coupled receptors, such as the Ca²⁺-sensing receptor (CaSR), have been identified throughout the brain, including hypothalamus. However, how the activation of CaSR in hypothalamic neurons and glial cells differentially modulate the intracellular Ca²⁺ ([Ca²⁺]_i) signaling, which could be important for functional crosstalk and downstream (pituitary) regulation, is unclear. Here, we first tested the activation of CaSR by extracellular Ca²⁺ ([Ca²⁺]_o) and L-arginine (allosteric agonist), in a neuro-glial co-culture model using mice hypothalamic neurons and glial cells to measure differential Ca²⁺ responses profile towards understanding their functional crosstalk. Compared to microglia and astrocytes, oligodendrocytes showed a steeper Ca²⁺ rise, but the increase in [Ca²⁺]_i in glial cells was distributed between the cellular compartments, except for in astrocytes. Ca²⁺ release of each neuronal component appeared to rise steadily, while the average Ca²⁺ release of cell bodies increased slightly at a sharper rate. Ca²⁺ entry of the cell body component was found to be most elevated compared to the axonal and dendritic components of the cell. With each dosage increase of L-arginine, the [Ca²⁺]_i levels of the neuron and oligodendrocytes were increased, with dendrites depicting the steepest increase compared to oligodendrocytes and the other neuronal components. [Ca²⁺]_i levels receded prior to implementing the fourth dosage (10&#xa0;mM), but were elevated again for dendrites, axons, and cell bodies, while response levels were mild for oligodendrocytes. Together these results reveal that the simultaneous activation of CaSR in neurons and glial cells could be used as an <i>in-vitro</i> tool towards understanding the physiology and pathophysiology of the central nervous system.</p>