ADENOSINE PRESYNAPTIC AND POSTSYNAPTIC MODULATION OF GLUTAMATE-DEPENDENT CALCIUM ACTIVITY IN HYPOTHALAMIC NEURONS

1. Within the hypothalamus, adenosine has been reported to influence t emperature regulation, sleep homeostasis, and endocrine secretions. Th e effects of adenosine on hypothalamic neurons have not been studied a t the cellular level. Adenosine (5 nM-30 mu M) showed no influence on intracellular Ca2+ or electrical activity in the presence of glutamate receptor antagonists D-2-amino-5-phosphonovalerate and 6-cyano-7-nitr oquinoxaline-2,3-dione; consequently, we examined the role of adenosin e in modulating the activity of glutamate in cultured hypothalamic neu rons (n > 1,700) with fura-2 Ca2+ digital imaging and whole cell patch -clamp electrophysiology in the absence of glutamate receptor block. 2 . When glutamate receptors were not blocked, adenosine (1-30 mu M) and the selective adenosine A(1) receptor agonist N-6-cyclopentyl adenosi ne (CPA; 5 nM-1 mu M) caused a large reduction in intracellular Ca2+ a nd electrical activity, suggesting that glutamate neurotransmission wa s critical for an effect of adenosine to be detected. Neuronal Ca2+ le vels were reversibly depressed by CPA (50 nM), with a maximum depressi on of 90%, and these effects were blocked by coadministration of the A (1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). 3. Ca2+ levels in immature neurons before the time of synaptogenesis wer e not affected by adenosine. Adenosine A(1) receptor activation suppre ssed glutamate-mediated Ca2+ activity in neurons in vitro 8 to 73 days . 4. Adenosine (1 or 10 mu M) caused a hyperpolarization of membrane p otential and a reduction of large postsynaptic potentials arising from endogenously released glutamate. The administration of low concentrat ions of CPA (5 nM) decreased the frequency of glutamate-mediated, neur onally synchronized Ca2+ transients and the frequency of postsynaptic potentials. 5. To compare the relative effects of adenosine on hypotha lamic neurons with cells from other brain regions, we assayed the effe cts of CPA on glutamate-mediated Ca2+ in hippocampal and cortical cult ures. CPA (50 nM) reversibly depressed glutamate-mediated Ca2+ rises i n hypothalamic neurons by 35%, compared with 54% in hippocampal neuron s and 46% in cortical neurons. 6. If it does play a functional role, a denosine should be released by hypothalamic cells. In some neurons the adenosine A(1) receptor antagonists cyclopentyltheophylline or DPCPX caused an increase in intracellular Ca2+, suggesting that adenosine wa s secreted by hypothalamic cells, tonically depressing glutamate-enhan ced neuronal Ca2+. 7. To determine whether adenosine could exert a pos tsynaptic effect, we coapplied it with glutamate agonists in the prese nce of tetrodotoxin. Within subpopulations of hypothalamic neurons, ad enosine and CPA either inhibited (18% of total neurons) or potentiated (6% of total neurons) responses to glutamate, N-methyl-D-aspartate, a nd kainate by greater than or equal to 20%. 8. In contrast to the mode st effects found in neurons, responses of hypothalamic astrocytes to t he application of glutamate or the metabotropic glutamate receptor ago nist (+/-)-trans-1-amino-1,3-cyclopentanedicarboxylic acid were strong ly potentiated by adenosine (mean +225%) and CPA. 9. Together, these f indings suggest that adenosine exerts a major presynaptic effect and a minor postsynaptic effect in the modulation of glutamate neurotransmi ssion in the hypothalamus, where it can play a significant role in blo cking a large part of the glutamate-induced Ca2+ rise. In the absence of glutamate transmission, adenosine has relatively little effect on e ither neuronal intracellular Ca2+ or electrical activity.