Neurotrophin-3 potentiates excitatory GABAergic synaptic transmission in cultured developing hypothalamic neurones of the rat.
1. Neurotrophin-3 (NT-3) supports the survival and differentiation of neurones in the central and peripheral nervous systems through a number of mechanisms that occur in a matter of hours or days. NT-3 may also have a more rapid mode of action that influences synaptic activity in mature neurones. In the present study, the effect of NT-3 on developing GABAergic synapses was investigated in 3- to 7-day-old cultures of rat hypothalamic neurones with whole-cell patch-clamp recording. 2. NT-3 induced a substantial dose-dependent potentiation of the frequency of spontaneous postsynaptic currents (sPSCs; 160 %) in developing neurones during a period when GABA evoked inward (depolarizing) current, as determined with gramicidin-perforated patch recordings. The NT-3 effect was long lasting; continued enhancement was found > 30 min after NT-3 wash-out. NT-3 evoked a substantial 202 % increase in total GABA-mediated inward current, measured as the time-current integral. Action potential frequency was also increased by NT-3 (to 220 %). 3. The frequency of GABA-mediated miniature postsynaptic currents in developing neurones in the presence of tetrodotoxin was potentiated (to 140%) by NT-3 with no change in the mean amplitude, suggesting a presynaptic locus of the effect. 4. In striking contrast to immature neurones, when more mature neurones were studied, NT-3 did not enhance the frequency of GABA-mediated spontaneous postsynaptic currents (sPSCs), but instead evoked a slight (16%) decrease. The frequency of miniature post-synaptic currents was also slightly decreased (16%) by the NT-3, with no change in amplitude. These results were recorded during a later period of neuronal maturity when GABA would evoke outward (hyperpolarizing) currents. NT-3 had no effect on the mean amplitude of GABA-evoked postsynaptic currents in either developing or mature neurones. 5. Intracellular application of K252a, a non-selective tyrosine kinase inhibitor, did not block the NT-3 effect postsynaptically. In contrast, bath application of K252a prevented the enhancement of sPSCs by NT-3, consistent with NT-3 acting through presynaptic induction of tyrosine kinase. Decreasing extracellular calcium with BAPTA or inhibiting calcium channels with Cd2+ blocked the augmentation of sPSC frequency by NT-3, suggesting that an increase of calcium entry may be required for the facilitation of NT-3. 6. Together, our results suggest NT-3 enhances GABA release during the developmental period when GABA is depolarizing and calcium elevating, but not later when GABA is inhibitory, suggesting that one mechanism through which NT-3 may influence neuronal development is via presynaptic potentiation of GABA excitation.
Gao
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Department of Neurosurgery, Yale University School of Medicine, New Haven, CT 06520, USA.
van den Pol
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AN
D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002227:Carbazoles / Q000494:pharmacology; D002478:Cells, Cultured; D004558:Electric Stimulation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects; D007031:Hypothalamus / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D026121:Indole Alkaloids; D008564:Membrane Potentials / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D020933:Neurotrophin 3 / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D013569:Synapses / Q000187:drug effects / Q000502:physiology; D009435:Synaptic Transmission / Q000187:drug effects*; D013779:Tetrodotoxin / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology / Q000502:physiology*
D002227:Carbazoles; D004791:Enzyme Inhibitors; D018691:Excitatory Amino Acid Antagonists; D026121:Indole Alkaloids; D020933:Neurotrophin 3; D013779:Tetrodotoxin; D005680:gamma-Aminobutyric Acid; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; C049985:staurosporine aglycone; D011505:Protein-Tyrosine Kinases; D002118:Calcium