Involvement of pre- and postsynaptic mechanisms in posttetanic potentiation at Aplysia synapses

Science

Volumn 275, Issue 5302, 1997, Pages 969-973

  Bao, Jian Xin ^a   Kandel, Eric R ^a   Hawkins, Robert D ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CALCIUM CHELATING AGENT; EGTAZIC ACID; ETHYLENE GLYCOL 1,2 BIS(2 AMINOPHENYL) ETHER N,N,N',N' TETRAACETIC ACID; OCTANOL;

ANIMAL CELL; APLYSIA; ARTICLE; CONTROLLED STUDY; EVOKED RESPONSE; EXCITATORY POSTSYNAPTIC POTENTIAL; HYPERPOLARIZATION; MOTONEURON; NERVE CELL CULTURE; NERVE CELL PLASTICITY; NONHUMAN; POST TETANIC POTENTIATION; PRIORITY JOURNAL; SENSORY NERVE CELL;

1-OCTANOL; ACTION POTENTIALS; ANIMALS; APLYSIA; CALCIUM; CELLS, CULTURED; CHELATING AGENTS; EGTAZIC ACID; LONG-TERM POTENTIATION; MOTOR NEURONS; NEURONAL PLASTICITY; NEURONS, AFFERENT; OCTANOLS; SEROTONIN; SYNAPSES; SYNAPTIC TRANSMISSION;

ANIMALIA; APLYSIA;

EID: 0031029077     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5302.969     Document Type: Article

References (47)

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14. _, ibid., p. 215.
15. A single identified L7 gill motor neuron or LFS siphon motor neuron was isolated from the abdominal ganglion of a juvenile (1 to 4 g) or adult (70 to 100 g) Aplysia respectively (Howard Hughes Medical Institute Mariculture Facility, Miami, FL), and co-cultured with a mechanosensory neuron isolated from the pleural ganglion of an adult animal. Several criteria were used to identify LFS cells [W. N. Frost, thesis, Columbia University, New York (1987), p. 113; L. S. Eliot, R. D. Hawkins, E. R. Kandel, S. Schacher, J. Neurosci. 14, 368 (1994)], although it is possible that some of the putative LFS cells were not motor neurons. The axon stump of the dissociated sensory neuron was placed near the initial segment of the main axon of the motor neuron, where functional synapses are formed. The cells were plated in culture dishes coated with poly-L-lysine (0.5 mg/ml) and left overnight at room temperature, after which the dishes were moved to an incubator where they were maintained at 18°C for 3 to 6 days. The culture medium consisted of 50% filtered Aplysia hemolymph and 50% L-15 medium (Flow Laboratories, McLean, VA) supplemented with salts (NaC1, 260 mM;
16. A single identified L7 gill motor neuron or LFS siphon motor neuron was isolated from the abdominal ganglion of a juvenile (1 to 4 g) or adult (70 to 100 g) Aplysia respectively (Howard Hughes Medical Institute Mariculture Facility, Miami, FL), and co-cultured with a mechanosensory neuron isolated from the pleural ganglion of an adult animal. Several criteria were used to identify LFS cells [W. N. Frost, thesis, Columbia University, New York (1987), p. 113; L. S. Eliot, R. D. Hawkins, E. R. Kandel, S. Schacher, J. Neurosci. 14, 368 (1994)], although it is possible that some of the putative LFS cells were not motor neurons. The axon stump of the dissociated sensory neuron was placed near the initial segment of the main axon of the motor neuron, where functional synapses are formed. The cells were plated in culture dishes coated with poly-L-lysine (0.5 mg/ml) and left overnight at room temperature, after which the dishes were moved to an incubator where they were maintained at 18°C for 3 to 6 days. The culture medium consisted of 50% filtered Aplysia hemolymph and 50% L-15 medium (Flow Laboratories, McLean, VA) supplemented with salts (NaC1, 260 mM;
17. 3, 2.3 mM) and D-glucose (34.6 mM). Additionally, glutamine (1 mM), penicillin (50 U/ml), and streptomycin (50 μg/ml) were also included in the culture medium. The hemolymph was collected from large animals (usually ∼1000 g) and stored at -70°C [S. Schacher and E. Proshansky, J. Neurosci. 3, 2403 (1983); S. Schacher, ibid. 5, 2028 (1985)].
18. 3, 2.3 mM) and D-glucose (34.6 mM). Additionally, glutamine (1 mM), penicillin (50 U/ml), and streptomycin (50 μg/ml) were also included in the culture medium. The hemolymph was collected from large animals (usually ∼1000 g) and stored at -70°C [S. Schacher and E. Proshansky, J. Neurosci. 3, 2403 (1983); S. Schacher, ibid. 5, 2028 (1985)].
19. 4, 27 mM). All chemicals were from Sigma (St. Louis, MO), except for BAPTA, which was from Molecular Probes (Eugene, OR).
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25. For EGTA injection into the sensory neurons, the tip of the injecting electrode was bevelled to have a resistance of 3 to 6 megohms. The electrodes were connected to a Pico-Injector (Medical Systems, Greenvale, NY) and pulses of pressure (800 ms duration; 0.3 to 1.4 psi) were delivered at 2-s intervals for 10 to 60 s. EGTA was first dissolved in KOH, and the pH was adjusted to 7.5 with HCl. Fast green (0.2%) was usually included in the EGTA solution or the control vehicle solution to visually ensure successful injection. The injection electrode was then withdrawn from the cell, and stimulation was begun with an extracellular electrode after a 30-min rest.
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27. The data in the text and figures are expressed as means ± SEM (n represents the number of cultures) and normalized to the first test EPSP (30 min after the impalement), except where otherwise indicated. Data were analyzed statistically either by t test (paired or unpaired) or by two-way analyses of variance (ANOVAs) with one repeated measure (time). If there was a significant overall effect in the ANOVA, comparisons were made at each time point by one-way ANOVAs, followed by Fisher PLSD tests. In the experiments shown in Fig. 1B, there were significant differences between control, PTP, and PTP after EGTA injection (PTP/EGTA) [F(2, 19) = 4.291 for treatment, P = 0.029; F(16, 152) = 5.894 for treatment × time, P = 0.0001]. There were no significant differences between the initial EPSPs or EPSPs 30 min after injection in the three groups shown in Fig. 1C.
28. 2+ concentration. In the experiments shown in Fig. 2, A and B, there were significant differences between homosynaptic depression (control), PTP, and PTP/ BAPTA [for LFS synapses: F(2, 18) = 6.281 for treatment, P = 0.0085; F(16, 144) = 2.428 for treatment × time, P = 0.0029; for L7 synapses: F(2, 18) = 6.214 for treatment, P = 0.0089; F(16, 144) = 7.191 for treatment × time, P = 0.0001]. For LFS synapses, the average amplitudes of the EPSPs on trial one, 30 min after impalement, were 26.4 ± 4.4 mV (control), 23.3 ± 3.7 mV (PTP), and 20.9 ± 3.3 mV (PTP/BAPTA); for L7 synapses, they were 17.3 ± 3.1 mV, 12.6 ± 3.2 mV, and 16.9 ± 3.9 mV, respectively. There were no significant differences among EPSP amplitudes on trial one for the three groups at either synapse.
29. We manually applied 10 μl of 5-HT (50 μM) with a 50-μl Hamilton syringe to the vicinity of the cells, as previously described (2). Fast green (0.2%), which by itself did not affect synaptic transmission, was included in the 5-HT solution to check the delivery and location of the puff. The solution was washed out in less than 60 s by continuous perfusion of the culture. A two-way ANOVA revealed that 5-HT produced significant facilitation of EPSPs [overall, F(2, 13) = 8.852 for treatment, P = 0.0037 and F(16, 104) = 5.718 for treatment × time, P = 0.0001], but there was no significant difference between the 5-HT and 5-HT/BAPTA groups. The average amplitudes of EPSPs 30 min after impalement were 25.3 ± 5.1 mV (control) and 19.3 ± 4.6 mV (BAPTA) in the experiments shown in Fig. 3B, and 22.7 ± 6.4 mV (control), 28.8 ± 3.6 mV (5-HT), and 20.1 ± 4.2 mV (5-HT/BAPTA) in the experiments shown in Fig. 3C. There were no significant differences among EPSP amplitudes on trial one for the different groups.
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32. LFS motor neurons were injected with 2 nA of current and L7 motor neurons with 4 nA, which produced approximately 50 to 100 mV of additional hyperpolarization below the level at which the motor neurons were held throughout the rest of the experiment (8), as measured in the soma. However, the hyperpolarization could have been substantially less at the synaptic region. Overall, there were significant differences between control, PTP, and PTP with hyperpolarization during the tetanus (PTP/HPP) [for LFS synapses: F(2, 14) = 6.771 for treatment, P = 0.0088; F(16, 112) = 4.807 for treatment × time, P = 0.0001; for L7 synapses: F(2, 19) = 5.22 for treatment, P = 0.0156; F(16, 152) = 2.951 for treatment × time, P = 0.0003]. For LFS synapses, EPSPs 30 min after impalement had amplitudes of 18.3 ± 2.5 mV (control), 21.3 ± 5.1 mV (PTP), and 22.1 ± 3.8 mV (PTP/HPP); for L7 synapses, they were 14.6 ± 2.6 mV, 19.5 ± 4.0 mV, and 18.1 ± 3.2 mV, respectively. There were no significant differences among EPSP amplitudes on trial one for these three groups.
33. Electrical coupling between the sensory neuron and motor neuron was monitored by impaling both cells with microelectrodes and applying a hyperpolarizing pulse (30 to 120 mV) to one cell (cell 1) and measuring the change of membrane potential in the other (cell 2). The degree of coupling was defined by: (membrane potential change in cell 2/hyperpolarizing potential applied in cell 1) × 100%. The coupling measured from soma to soma was 1.02 ± 0.49% (n = 5) from sensory to LFS cell, 0.79 ± 0.38% (n = 5) from LFS to sensory cell, 0.32 ± 0.17% (n = 8) from sensory to L7 cell, and 0.23 ± 0.12% (n = 8) from L7 to sensory cell.
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36. Overall, there were significant differences between control, PTP in the presence of octanol (PTP/octanol), and PTP with hyperpolarization during the tetanus in the presence of octanol [(PTP/HPP)/octanol] (F(2, 15) = 10.068 for treatment, P = 0.0176 and F(16, 132) = 4.594 for treatment × time, P = 0.0001]. EPSPs 30 min after impalement had amplitudes of 14.9 ± 2.3 mV (control), 11.6 ± 1.5 mV (PTP/octanol), and 12.0 ± 4.1 mV [(PTP/HPP)/octanol]. There were no significant differences among EPSP amplitudes on trial one for these three groups.
37. Recording of spontaneous mEPSPs was made at high gain, filtered at 300 Hz (-3 dB), and AC coupled. In experiments in which both evoked and spontaneous mEPSPs were measured, eEPSPs were recorded at low gain and DC coupled. Signals were digitized off-line with a PC-based ""fetchex" program (part of the ""pclamp" package, Axon Instruments) and analyzed with an ""axobasic" program. We visually identified mEPSPs on the basis of the following criteria: (i) a rise time of 3 to 20 ms, (ii) a half-decay time of at least 5 ms, and (iii) a minimum amplitude of 50 μV, which was usually about 25% greater than the peak-to-peak noise level (3 18). The frequency and the peak amplitude of mEPSPs were then automatically determined by the computer. When the dependent variable was the amplitude of eEPSPs (Fig. 4A), There were significant differences between the PTP and PTP/HPP groups [F(1, 10) = 5.244 for treatment, P = 0.045 and F(8, 80) = 6.189 for treatment × time, P = 0.001). EPSPs 30 min after impalement had amplitudes of 17.8 ± 2.9 mV (PTP) and 25.9 ± 2.3 mV (PTP/HPP) (not significantly different). When the dependent variable was the frequency of mEPSPs (Rg. 4B), there were no significant differences between PTP and PTP/HPP groups. The average frequency of mEPSPs 30 min after impalement was 0.032 ± 0.006 Hz and 0.041 ± 0.007 Hz for PTP and PTP/HPP groups, respectively (not significantly different).
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47. Supported by grants from the National Institute of Mental Health (MH 26212) and the Howard Hughes Medical Institute. A long-term fellowship from the Human Frontier Science Program Organization to J.-X.B. is gratefully acknowledged.