EMBRYONIC HYPOTHALAMIC EXPRESSION OF FUNCTIONAL GLUTAMATE RECEPTORS

Glutamate can play a number of roles in the developing brain, including modulation of gene expression, cell motility, neurite growth and neuronal survival, all critical for the final organization and function of the mature brain. These functions are dependent on the early expression of glutamate receptors and on glutamate release in developing neurons. This subject has received little attention in the hypothalamus, despite glutamate's critical role as an excitatory transmitter in hypothalamic control of circadian rhythms, endocrine secretion, temperature regulation, and autonomic control. A total of 10,922 rat hypothalamic neurons were studied with digital Ca2+ imaging with the ratiometric dye fura-2 to examine their responses to glutamate receptor agonists and antagonists during embryonic development and maturation in vitro. Functional glutamate receptors were found very early in development (embryonic day 15-E15) with both Ca2+ imaging and with patch damp recording. This is a time when the hypothalamus is beginning to undergo neurogenesis. Ca2+ responses from N-methyl-D-aspartate receptors developed later than those from non-N-methyl-D-aspartate ionotropic receptors that responded to kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazoleprionate. The responses of immature E15 cells after one day in vitro were compared with more mature cells after six days in vitro to examine the response to repeated 3 min applications of 100 mu M kainate (n = 108). Immature cells showed similar Ca2+ rises (+232 nM Ca2+) with each kainate stimulation. In contrast, more mature cells showed an initial Ca2+ rise of 307 nM, with the second rise only to 147 nM above the initial baseline. Immature cells more quickly returned to their pre-kainate baseline than did older cells. The expression of metabotropic glutamate receptors was studied with the selective agonist trans-1-amino-cyclopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five days in vitro, E16 astrocytes showed a greater response than did neurons to conditions that would activate the metabotropic glutamate receptor. A dramatic increase in the percentage of cells that responded to N-methyl-D-aspartate was found after only a few days in culture. Only a small number of E15 cells studied on the day of culture (4% of 694 cells) showed a response to 100 mu M N-methyl-D-aspartate. Thirty-eight percent of 120 E18 cells cultured for one day in vitro showed an N-methyl-D-aspartate response. By four days and vitro a 95% of 180 E18 cells with a neuronal morphology responded to N-methyl-D-aspartate. These results suggest that by the day of birth (after E22) almost all neurons probably have functional N-methyl-D-aspartate receptors. After five days in vitro, most neurons (78% of 192) decreased Ca2+ in response to glutamate ionotropic receptor antagonists DL-2-amino-5-phosphonopentanoic acid (100 mu M) and cyano-2, 3-dihydroxy-7-nitroquinoxaline (10 mu M). These data suggest that growing hypothalamic glutamatergic axons had already made functional synapses with the majority of neurons in the same culture dish by this time. In contrast, one day earlier only 16% of 192 neurons showed a Ca2+ decrease in response to glutamate receptor antagonists. Neurons (n = 46) were studied with whole cell patch clamp recording from E15, E17, and postnatal day 2 (P2) rats within a few hours of plating. In E15 neurons, inward current was seen in response to kainate (100 mu M), alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (30 mu M), N-methyl-D-aspartate (100 mu M), and glutamate (500 mu M), with 50% or more of the neurons showing responses to kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate, and glutamate; a smaller proportion responded to N-methyl-D-aspartate. Larger currents were evoked and a higher percentage of neurons (100%) responded to glutamate and its agonists if recordings were made from older P2 hypothalami. Even at the earliest age (E15), GABA (30 mu M) evoked large currents from all hypothalamic neurons examined (eight of eight). Taken together these data indicate that functional glutamate receptors are expressed early in hypothalamic embryonic development, at a time prior to synapse formation. At this early stage of development, glutamate induces intracellular Ca2+ increases sufficiently large to potentially influence many factors that play a role in neuronal development, including gene induction, neurite extension, enzyme regulation, and synaptogenesis.