A fundamental problem in the cellular analysis of learning and memory is the identification of the neuronal substrates of long-term information storage and their relation to short-term cellular alterations. In this report, biophysical correlates of long-term sensitization of a simple withdrawal reflex in the mollusc Aplysia were examined. A voltage-clamp analysis of the sensory neurons that control the reflex, 24 hours after sensitization training, revealed a significant reduction in net outward current. The results indicate that one mechanism for the storage of long-term sensitization is the regulation of membrane currents that influence the characteristics of the action potential and the excitability of individual neurons. The results also provide insights into the relation between short- and long-term sensitization in that the biophysical loci involved in the storage of long-term sensitization appear similar to those involved in short-term sensitization.
SUMMARY1. The effects of activation of GABAB receptors on Ca2+ currents (ICa) were investigated by application of whole-cell patch-clamp techniques to pyramidal neurones and non-pyramidal interneurones from the rat hippocampus grown in cell culture.2. (± )-Baclofen (10 /UM) reduced ICa evoked in pyramidal neurones at 0 mV from a holding potential of -80 mV by 33 + 3 %. Inhibition could be observed at the peak of ICa with significant inhibition still present after 200 ms at 0 mV. When Ba2+ was used as the charge carrier (IBa) baclofen inhibited 28 + 3 % of the current at -20 mV from a holding potential of -80 mV. The GABAB receptor antagonist 2-OHsaclofen (50-200 /lM) blocked the actions of baclofen.3. The selective Ca2+ channel blocker, w-conotoxin fraction GVIA (w-CgTX), was used to characterize the Ca21 currents inhibited by baclofen. w-CgTX (5/M) blocked 24 + 3 % of IBa. Following block of the wo-CgTX-sensitive current, baclofen inhibited significantly less current than under control conditions. 4. Addition of the dihydropyridine Ca2+ channel antagonist nimodipine (1 ,#M) inhibited 18 + 5 % of ICa at 0 mV from a holding potential of -80 mV and 44 + 9 % from a holding potential of -40 mV. In addition, nimodipine partially occluded subsequent responses to application of baclofen.5. In the presence of both 5 /,M-w-CgTX and 200 nM-nimodipine, responses to baclofen were almost completely blocked at depolarized holding potentials where the dihydropyridines are most effective.6. Inclusion of 500 /LM-guanosine 5'-O-(3-thiotriphosphate) (GTP-y-S) in the patch pipette enhanced the response to a subsaturating concentration of baclofen and rendered the response irreversible. Subsequent addition of the adenosine receptor agonist 2-Cl-adenosine (2-CA) (1 #tm; which also reduces ICa under control conditions) was without effect, suggesting that these two receptor-effector pathways converge.7. The actions of baclofen on ICa were blocked by pre-treatment of the cultures with pertussis toxin (250 ng/ml).
Excitatory synaptic transmission in the hippocampus involves the participation of at least two types of presynaptic Ca2+ channels, N-type channels sensitive to omega-conotoxin GVIA (omega-CTx GVIA) and Q-type channels sensitive to omega-agatoxin IVA (omega-Aga IVA). Hippocampal pyramidal neurons in cell culture were used to examine the participation of these two classes of channels at different stages of synapse development. Specific Ca2+ channel toxins were used to block presynaptic Ca2+ channels while whole-cell voltage-clamp recordings were used to record evoked EPSCs in postsynaptic neurons. At immature synapses (cells in culture for 10-15 d), omega-CTx GVIA (1-5 microM) blocked transmission by more than 80% while omega-Aga IVA (1 microM) was less effective. In older cultures, however, omega-Aga IVA (1 microM) was more effective than omega-CTx GVIA (1-5 microM) in blocking synaptic transmission. The pharmacological properties of the omega-Aga IVA sensitive component of synaptic transmission were examined in more detail using omega-Aga IVA and omega-conotoxin MVIIC (omega-CTx MVIIC). The properties of this component of transmitter release indicated that a Q-type Ca2+ channel was involved in presynaptic Ca2+ entry. The results suggest that different classes of presynaptic Ca2+ channels begin to participate in transmitter release at different times during synapse development and maturation.
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