Brain Research, 538 (1991) 347-350 Elsevier
347
BRES 24491
A test of the spine resistance hypothesis for LTP expression John Larson and Gary Lynch Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted 2 October 1990) Key words: Long-term potentiation; Dendritic spine; N-methyl-D-aspartatereceptor; Quisqualate receptor; Temperature; CA1; Hippocampus
Long-term potentiation (LTP) consists of an enhanced response to released transmitter by the quisqualate/AMPA subclass of glutamate receptors with little change in the slower currents generated by the NMDA receptor subclass. Recent computer simulations suggest that a decrease in the resistance of dendritic spines would selectively augment fast synaptic currents and this could produce the pattern of results found with LTP. The present experiments tested this hypothesis by asking whether non-NMDA responses slowed by low temperature to resemble NMDA responses could express LTP. Slow non-NMDA responses recorded at 25 °C did express LTP, indicating that the time courses of NMDA responses cannot explain why they do not express LTE The results, therefore, do not support the hypothesis that spine resistance changes are responsible for the enhanced transmission. Long-term potentiation (LTP) of synaptic transmission is a candidate mechanism for memory storage in the mammalian brain. It is well established that induction of LTP in field CA1 of hippocampus involves activation of N M D A (N-methyl-D-aspartate) receptors 6A° and a rise in calcium concentrations in the postsynaptic region2°'21; however the nature of the enduring substrate responsible for expression of the potentiation remains controversial. Electron microscopic studies have shown that the potentiation effect is associated with an increase in certain types of synapses but it is unclear if this reflects synaptogenesis or a transformation of existing connections 3'sA7As. LTP is expressed by postsynaptic responses to released transmitter that are mediated by receptors of the quisqualate/AMPA (Q/A) type but not of the N M D A type 12'22'23. This could indicate that LTP is due to a change in the properties of Q / A receptors or their associated ionophores. Receptor binding studies have thus far not provided evidence for changes in either number or affinity of Q/A receptors during LTP expression, but a number of technical factors could prevent detection of such a change 19. An alternative explanation for LTP expression is that a morphological alteration of dendritic spines increases current flow into the dendrite by decreasing the electrical resistance of the spine neck 2'7'13'24'26. The conditions under which spine shape could modify synaptic strength require both that the synaptic conductance be large enough and the spine neck conductance small enough that the synaptic current is attenuated by a large local
depolarization in the spine head 2'26. The degree of synaptic current attenuation could then be reduced by decreases in neck resistance as might occur with an increase in neck diameter or a decrease in neck length. Computer simulations of spines also show that slow synaptic currents are much less affected by neck resistance changes than are fast currents 26 and this could account for the relative lack of LTP expression by N M D A receptors, since NMDA-mediated responses are slower than Q/A-mediated responses 5'9't2'23. If this were the case, it would be expected that LTP could not be expressed by non-NMDA (Q/A) receptor-mediated responses under conditions in which they have time-courses similar to those generated by N M D A receptors that do not express LTE We tested this hypothesis in the present experiments by slowing Q/A responses with low temperatures until they resembled N M D A responses at physiological temperature. The results indicate that slow Q/A responses can express LTP and thus argue against the spine resistance hypothesis for LTP expression. Experiments were conducted on hippocampal slices maintained at the interface between a perfusion bath and a humidified, oxygen-rich (95% 02/5% CO2) atmosphere. Immediately after preparation, slices were placed in the recording chamber at a temperature of either 25 or 35 °C and maintained at the same temperature throughout the experiment. Slices were perfused (1 ml/min) with medium containing (in mM): 124 NaCl, 3 KCI, 1.25 KHEPO4, 2.5 MgSO4, 3.4 CaCI2, 10 D-glucose, 26 NaHCO3, and 3 L-ascorbate. Bipolar stimulation elec-
Correspondence: J. Larson, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717, U.S.A.
0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
348 trodes were placed in stratum radiatum of fields C A l a and C A l c to activate i n d e p e n d e n t inputs to a collection
were digitized at 10 kHz and measured by computer and
of CA1 cells; field potentials reflecting synaptic currents generated by the postsynaptic neurons were recorded
stored on disk. D N O X (6,7-dinitro-quinoxaline-2,3-dione) was obtained from Tocris Neuramin (Buckhurst Hill, U.K.) and D-AP5 (D-2-amino-5-phosphonopentanoate) was
with glass microelectrodes filled with 2 M NaCt (1-5 Mg2) •placed in stratum radiatum of C A l b . Evoked responses
purchased from Sigma (St. Louis, MO, U.S.A.). Evoked responses were recorded and quantified u n d e r 3 conditions; in all cases, stimulus intensity was set to evoke a field potential with a peak amplitude of about 1 mV. (a) Control responses mediated by O / A receptors at
......'.., wA~o
"........i .......
~ A ~ ' "..'""
NMDA
I 0 . s mv [ 5 mS
200
150 W O. 0 d
100 50 t0
TBS
lg
3b
4g
6b
75
me'
9b
TIME CMINUTES)
physiological temperature were measured in medium containing 2.5 mM M f + at 35 °C. (b) Responses mediated solely by N M D A receptors at physiological temperature (35 °C) were obtained by perfusing slices with Mg2+-free medium containing the n o n - N M D A receptor antagonist D N Q X . Baseline recordings were first taken in 2.5 mM Mg 2+ and then the perfusion solution was switched to one containing no added Mg 2+. D N Q X (20-40 MM) was either immediately added to this solution or applied 30-45 min later. N M D A receptormediated responses were quantified when the effect of D N Q X stabilized (about 30 min). After measurements were taken, the N M D A receptor antagonist D-AP5 (50 #M) was applied to verify that pure N M D A responses were quantified. (c) Slow Q/A receptor-mediated responses were measured in slices maintained at low temperature (25 °C) in medium containing 2.5 mM M f +. D-AP5 (50 #M) was used to test that the low temperature responses were mediated by n o n - N M D A
C
SECOND
FIRST
25'C
35* C
~
receptors. Theta burst stimulation (TBS) was used to elicit LTP and consisted of 10 high frequency bursts (4 pulses at 100 Hz) repeated at 5 Hz 16. Stimulus duration during TBS was 2-3 times that used to test the response. In LTP experiments, responses were tested at 20-s intervals for at
2 mV 20ms
Fig. 1. A: Q/A responses recorded at low temperature resemble NMDA responses recorded at physiological temperature. Shown are records of field potentials recorded under 3 conditions. Solid lines indicate responses mediated by Q/A receptors and NMDA receptors at 35 °C and the dotted line shows a Q/A receptor response at 25 °C. Note the similarity in time course of the low temperature Q/A response and the 35 °C NMDA response. B: Q/A receptor-mediated field potentials at low temperature express LTP. Temperature was maintained at 25 °C throughout the experiment. Graph shows measurements of the initial slope of the response (expressed as percent of the baseline average) before and after theta burst stimulation (TBS). TBS induced a stable synaptic potentiation of 45%. Records show typical field potentials immediately before and 1 h after TBS. C: responses to TBS are altered at low temperature. Recordings show field potentials evoked by the first and second of a series of 10 bursts given at 5 Hz in a slice maintained at 25 °C (top) and a slice maintained at 35 °C (bottom). At low temperature the enhancement of the second burst response is absent.
TABLE I
Mean field potential waveform parameters under different conditions Slope (mV/ms)
Amplitude (mV)
Risetime (ms)
Half-width (ms)
Control S.E.M. (n = 14)
0.431 0.025
1.001 0.030
4.43 0.19
6.84 0.37
NMDA S.E.M. (n = 14)
0.298 0.019
1.007 0.032
7.06 0.25
8.01 0.39
25 °C S.E.M. (n = 11)
0.305 0.021
1.056 0.020
6.32 0.20
8.94 0.27
Field potential slope was calculated as the maximum rate-of-rise, amplitude as peak amplitude, risetime as time-to-peak, and halfwidth as duration of response at half peak amplitude. Controls were recorded at 35 °C, NMDA responses at 35 °C in Mg2+-free medium and DNQX (20-40 ~M), 25 °C in 2.5 mM Mg2+ and no DNQX.
349 amplitude or temporal waveform of synaptic current in dendritic spines 26. Thus the effect of temperature on synaptic currents as measured by field potentials in the present experiments probably is not due to an increase in membrane resistance. A cooling-induced' decrease in conduction velocity of the afferent fibers probably occurs 4, but this should not affect the initial slope of the field potential unless it is accompanied by an increase in the variance of velocities. Cooling most likely slows the response by decreasing the speed of transmitter release and the operation of Q/A receptor-linked ion channels. Since both of these would have the same effect on the postsynaptic current, it is irrelevant to the spine resistance hypothesis how either contributes to the timecourse of the current. The main assumption involved in the present experiments is that the extracellular field potential faithfully reflects the waveform of the synaptic current. Theoretically, this should be the case 11 and experimental data suggest that the waveform of the field potential has a very similar shape to synaptic currents recorded from CA1 cells under voltage clamp conditions 1. In any case, it is difficult to imagine how the field potential could be slowed at low temperature without the synaptic current being similarly affected. The present results indicate that the temporal properties of currents carried by Q/A and N M D A receptors cannot explain why the former express LTP and the latter do not. Therefore, they do not support the spine resistance hypothesis for LTP expression. Alternative explanations include an alteration in the number or properties of Q/A receptors and associated ionophores or the formation of new synapses lacking N M D A receptors.
least 10 min prior to TBS and again for at least 30 min after TBS. As shown in Fig. 1A and Table I, NMDA responses were considerably slower than control responses mediated by Q/A receptors. However, by cooling slices to 25 °C, Q/A responses were evoked with time courses similar to N M D A responses at physiological temperature. Pure N M D A responses in MgE+-free medium and DNQX were completely antagonized by 50 tiM D-AP5 (n -- 14). Q/A responses at low temperature in 2.5 mM Mg 2+ were not affected by perfusion with the selective N M D A antagonist, D-AP5 (50/.tM, n = 10), indicating that they were mediated by Q/A receptors. Having obtained Q/A responses with waveforms similar to N M D A responses, we could test the spine resistance hypothesis for LTP expression. As shown in Fig. 1B, rhythmic 'theta burst' stimulation (TBS) can induce robust and stable LTP of Q/A responses at low temperature. In 7 of 11 experiments at 25 °C, stable LTP of at least 10% was induced (mean of 25.7% + 4.6% increase 20 rain after TBS, n = 11). The degree and probability of LTP was somewhat less than is normally seen at 35 °C, but analysis of the burst responses suggested that this was due to an impairment of the induction rather than expression of LTE As shown in Fig. 1C, slices at low temperature did not exhibit the characteristic enhancement of responses to bursts subsequent to the first burst seen at 35 °C. The burst response enhancement is partly due to a suppression of feedforward IPSPs, allowing enhanced summation within the burst and an activation of a current mediated by NMDA receptors; this current is required for LTP induction 14'15. Low temperature is reported to increase input resistance in these cells by about 75% over the range 37 to 27 °C (ref. 25) and we confirmed this (data not shown). Modeling studies indicate, however, that variations in membrane resistance have little effect on either the peak
This research was supported by grants from the Air Force Office of Scientific Research and the Office of Naval Research. We thank Dr. Charles J. Wilson for helpful discussion.
1 Barrionuevo, G., Kelso, S.R., Johnston, D. and Brown, T.H., Conductance mechanism responsible for long-term potentiation in monosynaptic and isolated excitatory synaptic inputs to hippocampus, J. Neurophysiol., 55 (1986) 540-550. 2 Brown,T.H., Chang, V.C., Ganong, A.J., Keenan, C.L. and Kelso, S.R., Biophysicalproperties of dendrites and spines that may control the induction and expression of long-term synaptic potentiation. In P.W. Landfield and S.A. Deadwyler (Eds.), Long-Term Potentiation: From Biophysics to Behavior, Alan R. Liss, New York, 1988, pp. 201-264. 3 Chang, E-L.F. and Greenough, W.T., Transient and enduring morphological correlates of synaptic activity and efficacychange m the rat hippocampal slice, Brain Research, 309 (1984) 35-46. 4 Chapman, R.A., Dependence on temperature of the conduction velocity of the action potential of the squid giant axon, Nature, 213 (1967) 1143-1144. 5 Collingridge, G.L., Herron, C.E. and Lester, R.A.J., Frequency dependent N-methyl-D-aspartate receptor-mediated synaptic transmission in rat hippocampus, J. Physiol., 399 (1988)
301-312. 6 Collingridge, G.L., Kehl, S.J. and McLennan, H., Excitatory amino acids in synaptic transmission in the Schaffer-commissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. 7 Coss, R.G. and Perkel, D.H., The function of dendritic spines: a review of theoretical issues, Behav. Neural. Biol., 44 (1985) 151-185. 8 Desmond, N.L. and Levy, W.B., Changes in the numerical density of synaptic contacts with long-term potentiation in the hippocampal dentate gyrus, J, Comp. Neurol., 253 (1986) 466-475. 9 Forsythe, I.D. and Westbrook, G.L., Slow excitatory postsynaptic currents mediated by N-methyl-D-aspartate receptors on cultured mouse central neurones, J. Physiol., 396 (1988) 515-533. 10 Harris, E.W., Ganong, A.H. and Cotman, C.W., Long-term potentiation in the hippocampus involves activation of Nmethyl-o-aspartate receptors, Brain Research, 323 (1984) 132137.
350 11 Hubbard, J.I., Llinas, R. and Quastel, D.M.J., Electrophysiological Analysis of Synaptic Transmission, Arnold, London, 1969. 12 Kauer, J.A., Malenka, R.C. and NicoU, R.A., A persistent postsynaptic modification mediates long-term potentiation in the hippocampus, Neuron, 1 (1988) 911-917. 13 Koch, C. and Poggio, T., A theoretical analysis of electrical properties of spines, Proc. Roy. Soc. Lond. B, 218 (1983) 455-477. 14 Larson, J. and Lynch, G., Induction of synaptic potentiation in hippocampus by patterned stimulation involves two events, Science, 232 (1986) 985-988. 15 Larson, J. and Lynch, G., Role of N-methyl-D-aspartate receptors in the induction of synaptic potentiation by burst stimulation patterned after the hippocampal theta rhythm, Brain Research, 441 (1988) 111-118. 16 Larson, J., Wong, D. and Lynch, G., Patterned stimulation at the theta frequency is optimal for the induction of hippocampal long-term potentiation, Brain Research, 368 (1986) 347-350. 17 Lee, K., Oliver, M., Schottler, E and Lynch, G., Electron microscopic studies of brain slices: the effects of high frequency stimulation on dendritic ultrastructure. In G. Kerkut and H.V. Wheal (Eds.), Electrical Activity in Isolated Mammalian C.N.S. Preparations, Academic Press, New York, 1981, pp. 189-212. 18 Lee, K., Schottler, E, Oliver, M. and Lynch, G., Brief bursts of high frequency stimulation produce two types of structural changes in rat hippocampus, J. Neurophysiol., 44 (1980) 247258.
19 Lynch, G., Kessler, M., Arai, A. and Larson, J., The nature and causes of hippocampal long-term potentiation. In J. StormMathisen, J. Zimmer and O.P. Ottersen (Eds.), Progress in Brain Research. Vol. 83. Understanding the Brain through the Hippocampus: The Hippocampal Region as a Model for Studying Brain Structure and Function, Elsevier Science Publishers, Amsterdam, 1990, pp. 233-250. 20 Lynch, G., Larson, J., Kelso, S., Barrionuevo, G. and Schottler, F., Intracellular injections of EGTA block induction of hippocampal long-term potentiation, Nature, 305 (1983) 719-721. 21 Malenka, R.C., Kauer, J.A., Zucker, R.S. and Nicoll, R.A., Postsynaptic calcium is sufficient for potentiation of hippocampal synaptic transmission, Science, 242 (1988) 8t-84. 22 Muller, D. and Lynch, G., Long-term potentiation differentially affects two components of synaptic responses in hippocampus, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 9346-9350. 23 Muller, D., Joly, M. and Lynch, G., Contributions of quisqualate and NMDA receptors to the induction and expression of LTP, Science, 242 (1988) 1694-1697. 24 Rail, W., Dendritic spines and synaptic potency. In R. Porter (Ed.), Studies in Neurophysiology, Cambridge Univ. Press, Cambridge, 1978, pp. 203-209. 25 Thompson, S.M., Masukawa, L.M. and Prince, D.A., Temperature dependence of intrinsic membrane properties and synaptic potentials in hippocampal CA1 neurons in vitro, J. Neurosci., 5 (1985) 817-824. 26 Wilson, C.J., Passive cable properties of dendritic spines and spiny neurons, J. Neurosci., 4 (1984) 281-297.
347
BRES 24491
A test of the spine resistance hypothesis for LTP expression John Larson and Gary Lynch Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717 (U.S.A.)
(Accepted 2 October 1990) Key words: Long-term potentiation; Dendritic spine; N-methyl-D-aspartatereceptor; Quisqualate receptor; Temperature; CA1; Hippocampus
Long-term potentiation (LTP) consists of an enhanced response to released transmitter by the quisqualate/AMPA subclass of glutamate receptors with little change in the slower currents generated by the NMDA receptor subclass. Recent computer simulations suggest that a decrease in the resistance of dendritic spines would selectively augment fast synaptic currents and this could produce the pattern of results found with LTP. The present experiments tested this hypothesis by asking whether non-NMDA responses slowed by low temperature to resemble NMDA responses could express LTP. Slow non-NMDA responses recorded at 25 °C did express LTP, indicating that the time courses of NMDA responses cannot explain why they do not express LTE The results, therefore, do not support the hypothesis that spine resistance changes are responsible for the enhanced transmission. Long-term potentiation (LTP) of synaptic transmission is a candidate mechanism for memory storage in the mammalian brain. It is well established that induction of LTP in field CA1 of hippocampus involves activation of N M D A (N-methyl-D-aspartate) receptors 6A° and a rise in calcium concentrations in the postsynaptic region2°'21; however the nature of the enduring substrate responsible for expression of the potentiation remains controversial. Electron microscopic studies have shown that the potentiation effect is associated with an increase in certain types of synapses but it is unclear if this reflects synaptogenesis or a transformation of existing connections 3'sA7As. LTP is expressed by postsynaptic responses to released transmitter that are mediated by receptors of the quisqualate/AMPA (Q/A) type but not of the N M D A type 12'22'23. This could indicate that LTP is due to a change in the properties of Q / A receptors or their associated ionophores. Receptor binding studies have thus far not provided evidence for changes in either number or affinity of Q/A receptors during LTP expression, but a number of technical factors could prevent detection of such a change 19. An alternative explanation for LTP expression is that a morphological alteration of dendritic spines increases current flow into the dendrite by decreasing the electrical resistance of the spine neck 2'7'13'24'26. The conditions under which spine shape could modify synaptic strength require both that the synaptic conductance be large enough and the spine neck conductance small enough that the synaptic current is attenuated by a large local
depolarization in the spine head 2'26. The degree of synaptic current attenuation could then be reduced by decreases in neck resistance as might occur with an increase in neck diameter or a decrease in neck length. Computer simulations of spines also show that slow synaptic currents are much less affected by neck resistance changes than are fast currents 26 and this could account for the relative lack of LTP expression by N M D A receptors, since NMDA-mediated responses are slower than Q/A-mediated responses 5'9't2'23. If this were the case, it would be expected that LTP could not be expressed by non-NMDA (Q/A) receptor-mediated responses under conditions in which they have time-courses similar to those generated by N M D A receptors that do not express LTE We tested this hypothesis in the present experiments by slowing Q/A responses with low temperatures until they resembled N M D A responses at physiological temperature. The results indicate that slow Q/A responses can express LTP and thus argue against the spine resistance hypothesis for LTP expression. Experiments were conducted on hippocampal slices maintained at the interface between a perfusion bath and a humidified, oxygen-rich (95% 02/5% CO2) atmosphere. Immediately after preparation, slices were placed in the recording chamber at a temperature of either 25 or 35 °C and maintained at the same temperature throughout the experiment. Slices were perfused (1 ml/min) with medium containing (in mM): 124 NaCl, 3 KCI, 1.25 KHEPO4, 2.5 MgSO4, 3.4 CaCI2, 10 D-glucose, 26 NaHCO3, and 3 L-ascorbate. Bipolar stimulation elec-
Correspondence: J. Larson, Center for the Neurobiology of Learning and Memory, University of California, Irvine, CA 92717, U.S.A.
0006-8993/91/$03.50 (~) 1991 Elsevier Science Publishers B.V. (Biomedical Division)
348 trodes were placed in stratum radiatum of fields C A l a and C A l c to activate i n d e p e n d e n t inputs to a collection
were digitized at 10 kHz and measured by computer and
of CA1 cells; field potentials reflecting synaptic currents generated by the postsynaptic neurons were recorded
stored on disk. D N O X (6,7-dinitro-quinoxaline-2,3-dione) was obtained from Tocris Neuramin (Buckhurst Hill, U.K.) and D-AP5 (D-2-amino-5-phosphonopentanoate) was
with glass microelectrodes filled with 2 M NaCt (1-5 Mg2) •placed in stratum radiatum of C A l b . Evoked responses
purchased from Sigma (St. Louis, MO, U.S.A.). Evoked responses were recorded and quantified u n d e r 3 conditions; in all cases, stimulus intensity was set to evoke a field potential with a peak amplitude of about 1 mV. (a) Control responses mediated by O / A receptors at
......'.., wA~o
"........i .......
~ A ~ ' "..'""
NMDA
I 0 . s mv [ 5 mS
200
150 W O. 0 d
100 50 t0
TBS
lg
3b
4g
6b
75
me'
9b
TIME CMINUTES)
physiological temperature were measured in medium containing 2.5 mM M f + at 35 °C. (b) Responses mediated solely by N M D A receptors at physiological temperature (35 °C) were obtained by perfusing slices with Mg2+-free medium containing the n o n - N M D A receptor antagonist D N Q X . Baseline recordings were first taken in 2.5 mM Mg 2+ and then the perfusion solution was switched to one containing no added Mg 2+. D N Q X (20-40 MM) was either immediately added to this solution or applied 30-45 min later. N M D A receptormediated responses were quantified when the effect of D N Q X stabilized (about 30 min). After measurements were taken, the N M D A receptor antagonist D-AP5 (50 #M) was applied to verify that pure N M D A responses were quantified. (c) Slow Q/A receptor-mediated responses were measured in slices maintained at low temperature (25 °C) in medium containing 2.5 mM M f +. D-AP5 (50 #M) was used to test that the low temperature responses were mediated by n o n - N M D A
C
SECOND
FIRST
25'C
35* C
~
receptors. Theta burst stimulation (TBS) was used to elicit LTP and consisted of 10 high frequency bursts (4 pulses at 100 Hz) repeated at 5 Hz 16. Stimulus duration during TBS was 2-3 times that used to test the response. In LTP experiments, responses were tested at 20-s intervals for at
2 mV 20ms
Fig. 1. A: Q/A responses recorded at low temperature resemble NMDA responses recorded at physiological temperature. Shown are records of field potentials recorded under 3 conditions. Solid lines indicate responses mediated by Q/A receptors and NMDA receptors at 35 °C and the dotted line shows a Q/A receptor response at 25 °C. Note the similarity in time course of the low temperature Q/A response and the 35 °C NMDA response. B: Q/A receptor-mediated field potentials at low temperature express LTP. Temperature was maintained at 25 °C throughout the experiment. Graph shows measurements of the initial slope of the response (expressed as percent of the baseline average) before and after theta burst stimulation (TBS). TBS induced a stable synaptic potentiation of 45%. Records show typical field potentials immediately before and 1 h after TBS. C: responses to TBS are altered at low temperature. Recordings show field potentials evoked by the first and second of a series of 10 bursts given at 5 Hz in a slice maintained at 25 °C (top) and a slice maintained at 35 °C (bottom). At low temperature the enhancement of the second burst response is absent.
TABLE I
Mean field potential waveform parameters under different conditions Slope (mV/ms)
Amplitude (mV)
Risetime (ms)
Half-width (ms)
Control S.E.M. (n = 14)
0.431 0.025
1.001 0.030
4.43 0.19
6.84 0.37
NMDA S.E.M. (n = 14)
0.298 0.019
1.007 0.032
7.06 0.25
8.01 0.39
25 °C S.E.M. (n = 11)
0.305 0.021
1.056 0.020
6.32 0.20
8.94 0.27
Field potential slope was calculated as the maximum rate-of-rise, amplitude as peak amplitude, risetime as time-to-peak, and halfwidth as duration of response at half peak amplitude. Controls were recorded at 35 °C, NMDA responses at 35 °C in Mg2+-free medium and DNQX (20-40 ~M), 25 °C in 2.5 mM Mg2+ and no DNQX.
349 amplitude or temporal waveform of synaptic current in dendritic spines 26. Thus the effect of temperature on synaptic currents as measured by field potentials in the present experiments probably is not due to an increase in membrane resistance. A cooling-induced' decrease in conduction velocity of the afferent fibers probably occurs 4, but this should not affect the initial slope of the field potential unless it is accompanied by an increase in the variance of velocities. Cooling most likely slows the response by decreasing the speed of transmitter release and the operation of Q/A receptor-linked ion channels. Since both of these would have the same effect on the postsynaptic current, it is irrelevant to the spine resistance hypothesis how either contributes to the timecourse of the current. The main assumption involved in the present experiments is that the extracellular field potential faithfully reflects the waveform of the synaptic current. Theoretically, this should be the case 11 and experimental data suggest that the waveform of the field potential has a very similar shape to synaptic currents recorded from CA1 cells under voltage clamp conditions 1. In any case, it is difficult to imagine how the field potential could be slowed at low temperature without the synaptic current being similarly affected. The present results indicate that the temporal properties of currents carried by Q/A and N M D A receptors cannot explain why the former express LTP and the latter do not. Therefore, they do not support the spine resistance hypothesis for LTP expression. Alternative explanations include an alteration in the number or properties of Q/A receptors and associated ionophores or the formation of new synapses lacking N M D A receptors.
least 10 min prior to TBS and again for at least 30 min after TBS. As shown in Fig. 1A and Table I, NMDA responses were considerably slower than control responses mediated by Q/A receptors. However, by cooling slices to 25 °C, Q/A responses were evoked with time courses similar to N M D A responses at physiological temperature. Pure N M D A responses in MgE+-free medium and DNQX were completely antagonized by 50 tiM D-AP5 (n -- 14). Q/A responses at low temperature in 2.5 mM Mg 2+ were not affected by perfusion with the selective N M D A antagonist, D-AP5 (50/.tM, n = 10), indicating that they were mediated by Q/A receptors. Having obtained Q/A responses with waveforms similar to N M D A responses, we could test the spine resistance hypothesis for LTP expression. As shown in Fig. 1B, rhythmic 'theta burst' stimulation (TBS) can induce robust and stable LTP of Q/A responses at low temperature. In 7 of 11 experiments at 25 °C, stable LTP of at least 10% was induced (mean of 25.7% + 4.6% increase 20 rain after TBS, n = 11). The degree and probability of LTP was somewhat less than is normally seen at 35 °C, but analysis of the burst responses suggested that this was due to an impairment of the induction rather than expression of LTE As shown in Fig. 1C, slices at low temperature did not exhibit the characteristic enhancement of responses to bursts subsequent to the first burst seen at 35 °C. The burst response enhancement is partly due to a suppression of feedforward IPSPs, allowing enhanced summation within the burst and an activation of a current mediated by NMDA receptors; this current is required for LTP induction 14'15. Low temperature is reported to increase input resistance in these cells by about 75% over the range 37 to 27 °C (ref. 25) and we confirmed this (data not shown). Modeling studies indicate, however, that variations in membrane resistance have little effect on either the peak
This research was supported by grants from the Air Force Office of Scientific Research and the Office of Naval Research. We thank Dr. Charles J. Wilson for helpful discussion.
1 Barrionuevo, G., Kelso, S.R., Johnston, D. and Brown, T.H., Conductance mechanism responsible for long-term potentiation in monosynaptic and isolated excitatory synaptic inputs to hippocampus, J. Neurophysiol., 55 (1986) 540-550. 2 Brown,T.H., Chang, V.C., Ganong, A.J., Keenan, C.L. and Kelso, S.R., Biophysicalproperties of dendrites and spines that may control the induction and expression of long-term synaptic potentiation. In P.W. Landfield and S.A. Deadwyler (Eds.), Long-Term Potentiation: From Biophysics to Behavior, Alan R. Liss, New York, 1988, pp. 201-264. 3 Chang, E-L.F. and Greenough, W.T., Transient and enduring morphological correlates of synaptic activity and efficacychange m the rat hippocampal slice, Brain Research, 309 (1984) 35-46. 4 Chapman, R.A., Dependence on temperature of the conduction velocity of the action potential of the squid giant axon, Nature, 213 (1967) 1143-1144. 5 Collingridge, G.L., Herron, C.E. and Lester, R.A.J., Frequency dependent N-methyl-D-aspartate receptor-mediated synaptic transmission in rat hippocampus, J. Physiol., 399 (1988)
301-312. 6 Collingridge, G.L., Kehl, S.J. and McLennan, H., Excitatory amino acids in synaptic transmission in the Schaffer-commissural pathway of the rat hippocampus, J. Physiol., 334 (1983) 33-46. 7 Coss, R.G. and Perkel, D.H., The function of dendritic spines: a review of theoretical issues, Behav. Neural. Biol., 44 (1985) 151-185. 8 Desmond, N.L. and Levy, W.B., Changes in the numerical density of synaptic contacts with long-term potentiation in the hippocampal dentate gyrus, J, Comp. Neurol., 253 (1986) 466-475. 9 Forsythe, I.D. and Westbrook, G.L., Slow excitatory postsynaptic currents mediated by N-methyl-D-aspartate receptors on cultured mouse central neurones, J. Physiol., 396 (1988) 515-533. 10 Harris, E.W., Ganong, A.H. and Cotman, C.W., Long-term potentiation in the hippocampus involves activation of Nmethyl-o-aspartate receptors, Brain Research, 323 (1984) 132137.
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