EMBRYONIC HYPOTHALAMIC EXPRESSION OF FUNCTIONAL GLUTAMATE RECEPTORS

Glutamate can play a number of roles in the developing brain, includin g modulation of gene expression, cell motility, neurite growth and neu ronal survival, all critical for the final organization and function o f the mature brain. These functions are dependent on the early express ion of glutamate receptors and on glutamate release in developing neur ons. This subject has received little attention in the hypothalamus, d espite glutamate's critical role as an excitatory transmitter in hypot halamic 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 d ye fura-2 to examine their responses to glutamate receptor agonists an d antagonists during embryonic development and maturation in vitro. Fu nctional glutamate receptors were found very early in development (emb ryonic day 15-E15) with both Ca2+ imaging and with patch damp recordin g. This is a time when the hypothalamus is beginning to undergo neurog enesis. Ca2+ responses from N-methyl-D-aspartate receptors developed l ater than those from non-N-methyl-D-aspartate ionotropic receptors tha t responded to kainate and lpha-amino-3-hydroxy-5-methyl-4-isoxazolepr ionate. The responses of immature E15 cells after one day in vitro wer e compared with more mature cells after six days in vitro to examine t he response to repeated 3 min applications of 100 mu M kainate (n = 10 8). 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 ini tial baseline. Immature cells more quickly returned to their pre-kaina te baseline than did older cells. The expression of metabotropic gluta mate receptors was studied with the selective agonist trans-1-amino-cy clopentyl-1,3-dicarboxylic acid and with glutamate stimulation in the absence of extracellular Ca2+ and presence of 1 mM EGTA. After five da ys 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-m ethyl-D-aspartate was found after only a few days in culture. Only a s mall number of E15 cells studied on the day of culture (4% of 694 cell s) showed a response to 100 mu M N-methyl-D-aspartate. Thirty-eight pe rcent of 120 E18 cells cultured for one day in vitro showed an N-methy l-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 re sults suggest that by the day of birth (after E22) almost all neurons probably have functional N-methyl-D-aspartate receptors. After five da ys in vitro, most neurons (78% of 192) decreased Ca2+ in response to g lutamate ionotropic receptor antagonists DL-2-amino-5-phosphonopentano ic acid (100 mu M) and cyano-2, 3-dihydroxy-7-nitroquinoxaline (10 mu M). These data suggest that growing hypothalamic glutamatergic axons h ad already made functional synapses with the majority of neurons in th e same culture dish by this time. In contrast, one day earlier only 16 % of 192 neurons showed a Ca2+ decrease in response to glutamate recep tor antagonists. Neurons (n = 46) were studied with whole cell patch c lamp recording from E15, E17, and postnatal day 2 (P2) rats within a f ew hours of plating. In E15 neurons, inward current was seen in respon se to kainate (100 mu M), ha-amino-3-hydroxy-5-methyl-4-isoxazolepropr ionate (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, a -amino-3-hydroxy-5-methyl-4-isoxazoleproprionate, and glutamate; a sma ller proportion responded to N-methyl-D-aspartate. Larger currents wer e evoked and a higher percentage of neurons (100%) responded to glutam ate 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 togeth er these data indicate that functional glutamate receptors are express ed early in hypothalamic embryonic development, at a time prior to syn apse formation. At this early stage of development, glutamate induces intracellular Ca2+ increases sufficiently large to potentially influen ce many factors that play a role in neuronal development, including ge ne induction, neurite extension, enzyme regulation, and synaptogenesis .