Article Figures & Data
Figures
- Figure 1.
Learning resulted in significantly reduced postburst AHP evoked with direct current injections into the soma of CA1 pyramidal neurons. A, Rats were trained twice a day for 3 d, and electrophysiological recordings were made on the fourth day. B, Conditioned rats (closed circles; N = 26) exhibited significantly more adaptive responses compared with pseudoconditioned rats (gray circles; N = 21) across the five training sessions (rmANOVA: F(1,49) = 123.8, p < 0.0001; *unpaired t test p < 0.01). C, D, Examples of integrated EMG response are illustrated from a conditioned (C) and a pseudoconditioned (D) rat. C illustrates an adaptive response to a paired presentation of the tone (CS) and airpuff (US) during session 5. D illustrates the lack of an adaptive response to a CS-alone trial and an US-alone trial from a pseudoconditioned rat during session 5. E, Example of postburst AHP traces evoked with direct current injections into CA1 pyramidal neurons from a conditioned and a pseudoconditioned rat. Calibration: 4 mV, 500 ms. F, The postburst AHP evoked using direct somatic current injections was significantly reduced in CA1 pyramidal neurons from rats that had learned the task as compared with those from pseudo and naive rats (F(2,87) = 12.98, p < 0.0001; Fisher's PLSD p < 0.05 conditioned vs pseudo and naive). Smaller circles are the data points from individual neurons. Larger circles are the group means. Error bars are SEM (obscured by the group mean circles). Open circle, Naive; gray circle, pseudoconditioned; black circle, conditioned. n: naive 32, pseudo 26, conditioned 32.
- Figure 2.
Learning resulted in postburst AHP reduction and IPSP increase in CA1 pyramidal neurons. A, Example postburst hyperpolarization traces evoked with 50 Hz train of five suprathreshold Schaffer collateral stimulation from a CA1 neuron from a conditioned and a pseudoconditioned rats before (Pre) and after (Post) GABA blockers. The action potentials have been truncated to focus on the hyperpolarization. Calibration: 4 mV, 500 ms. B, Repeated ANOVA revealed significant training (F(2,26) = 4.82, p < 0.05) and drug (F(1,26) = 79.26, p < 0.0001) effects. Further analysis revealed no significant difference in the postburst hyperpolarization in normal aCSF (pre-GABA ANOVA; F(2,26) = 0.05, p > 0.95). GABA blockers significantly reduced the hyperpolarization in CA1 neurons from all rats (#paired t test p < 0.05) and unveiled the learning-related postburst AHP reduction in neurons from conditioned rats (post-GABA ANOVA; F(2,26) = 5.77, p < 0.01; *Fisher's PLSD p < 0.05). Open circles, Pre-GABA blockers; closed circles, post-GABA blockers. n: naive 10, pseudo 9, conditioned 10. C, Example traces of hyperpolarization evoked with single synaptically evoked action potential from CA1 neurons from conditioned and pseudoconditioned rats before (Pre) and after (Post) GABA blockers. Calibration: 5 mV, 100 ms. D, Repeated ANOVA revealed significant drug (F(1,33) = 114.27, p < 0.0001) and interaction of drug and training (F(2,33) = 9.51, p = 0.0005) effects on the IPSP following a single synaptically evoked AP. Further analysis revealed that GABA blockers significantly reduced the IPSP in CA1 neurons from all behavioral groups (#paired t test p < 0.05) and abolished the learning-related IPSP enlargement observed in normal aCSF (pre-GABA ANOVA: F(2,33) = 6.88, p < 0.005; *Fisher's PLSD p < 0.05 conditioned vs pseudo and naive; post-GABA ANOVA: F(2,33) = 1.35, p > 0.25). n: naive 13, pseudo 10, conditioned 13. E, Illustrated are example traces evoked with 50 Hz trains of five subthreshold Schaffer collateral stimulation in CA1 neurons from conditioned and pseudoconditioned rats before (Pre) and after (Post) GABA blockers. Calibration: 3 mV, 100 ms. F, Repeated ANOVA revealed significant drug (F(1,29) = 6.88, p < 0.005) and interaction of drug and training (F(2,29) = 4.66, p < 0.05) effects on the IPSP following a 50 Hz train of subthreshold EPSPs. Further analysis revealed that GABA blockers significantly reduced the IPSP in CA1 neurons from all behavioral groups (#paired t test p < 0.05). No significant IPSP differences were observed between groups either before or after GABA blockers. n: naive 9, pseudo 9, conditioned 11. G–J, IPSPs and EPSPs were evoked using fixed voltage stimulation with a Digitimer-isolated stimulator and a stimulating electrode placed in the stratum radiatum. G, Illustrated is an example of the evoked IPSPs and the resulting input/output slope determined by a linear regression of the IPSP amplitude and the fixed voltage stimulation. Calibration: 100 ms, 1 mV. H, J, No significant differences in the input/output slopes were observed for either the IPSP or the EPSP between the training groups (naive—white, n = 13; pseudo—gray, n = 9; conditioned—black, n = 14). I, Illustrated is an example of the evoked EPSPs in the presence of GABA blockers and the resulting input/output slope determined by a linear regression of the EPSP amplitude and the fixed voltage stimulation. Calibration: 100 ms, 5 mV.
- Figure 3.
The frequency of spontaneous and miniature IPSCs is increased onto pyramidal neurons from conditioned rats compared with controls. A, The frequency, but not amplitude, of sIPSCs is increased onto CA1 pyramidal neurons of conditioned rats (closed circles) compared with naive (clear circles) and pseudoconditioned rats (gray circles). n: naive 20, pseudo 27, conditioned 22. Calibration: 20 pA, 250 ms. B, The frequency, but not amplitude, of mIPSCs (recorded in the presence of 100 nm TTX) is increased onto CA1 pyramidal neurons of conditioned rats compared with naive and pseudoconditioned rats. Calibration: 20 pA, 250 ms. n: naive 18, pseudo 19, conditioned 19. Note: *Fischer's PLSD p < 0.05. Smaller circles are the data points from individual neurons. Larger circles are the group means. In some instances, the SEM of each group is smaller than the size of the circle that represents the group mean data.
- Figure 4.
Intrinsic excitability is increased in SOMs from trained mice compared with controls. A, Mice were trained twice a day for 3 d, and electrophysiological recordings were made on the fourth day. B, Conditioned mice (closed circles; N = 29) exhibited significantly more adaptive responses compared with pseudoconditioned mice (open circles; N = 24) (rmANOVA: F(1,51) = 117.4, p < 0.0001; *unpaired t test p < 0.01). C, D, Examples of integrated EMG response are illustrated from a conditioned (C) and a pseudoconditioned (D) mouse. C illustrates an adaptive response to a paired presentation of the whisker vibration (CS) and periorbital shock (US) during session 5. D illustrates the lack of an adaptive response to a CS-alone trial and an US-alone trial from a pseudoconditioned mouse during session 5. E, Learning resulted in significantly increased spontaneous firing rate at resting membrane potential in SOM neurons. Conditioned, black circle; pseudoconditioned, gray circle; naive, open circle. Calibration: 10 mV, 500 ms. F, The AHP following a single action potential is significantly reduced in SOMs from conditioned mice compared with controls. Calibration: 10 mV, 50 ms. G, The current required to elicit 20 action potentials in 1 s was significantly less in cells from conditioned animals compared with controls. Calibration: 20 mV, 150 pA, 200 ms. n: naive 25, pseudo 32, conditioned 32. Note: *Fischer's PLSD p < 0.05. Smaller circles are the data points from individual neurons. Larger circles are the group means. In some instances, the SEM of each group is smaller than the size of the circle that represents the group mean data.
- Figure 5.
Learning-related increased intrinsic excitability of SOM neurons is mediated by reductions in the apamin-sensitive AHP. A, Repeated ANOVA revealed significant training (F(2,25) = 5.96, p < 0.001) and apamin (F(1,25) = 3.84, p < 0.05) effects on the peak AHP. Before apamin application, the peak AHP was significantly reduced in SOM neurons from conditioned mice (pre-apamin ANOVA: F(2,25) = 5.77, p < 0.01; *Fischer's PLSD p < 0.05). Apamin (500 nm) reduced the AHP following a single action potential in cells from pseudoconditioned and naive animals, but not from conditioned animals (#paired t tests p < 0.05). After apamin there was no difference between groups in the size of the AHP. B, Repeated ANOVA revealed significant training (F(2,25) = 5.76, p < 0.01) and apamin (F(1,25) = 4.17, p < 0.05) effects on the current necessary to elicit 20 action potentials (APs) in 1 s. Before apamin application, significantly less current was needed in SOM neurons from conditioned mice to elicit 20 APs (pre-apamin ANOVA: F(2,25) = 4.00, p < 0.05; *Fischer's PLSD p < 0.05). Apamin (500 nm) decreased the amount of current required to elicit 20 APs in cells from pseudoconditioned and naive animals, but not from conditioned animals (#paired t tests p < 0.05). After apamin, there was no difference between groups in the degree of accommodation. n: naive 9, pseudo 10, conditioned 10. C, Repeated ANOVA revealed significant training (F(2,25) = 4.22, p < 0.05) and NS309 (F(1,25) = 4.59, p < 0.05) effects on the peak AHP. Before NS309 application, the peak AHP was significantly reduced in SOM neurons from conditioned mice (pre-NS309 ANOVA: F(2,25) = 3.45, p < 0.05; *Fischer's PLSD p < 0.05). Application of 10 μm NS309 increased the size of the AHP in neurons from all training groups (#paired t tests p < 0.005) and abolished the learning-related AHP reduction. D, Repeated ANOVA revealed significant training (F(2,27) = 4.90, p < 0.05) and NS309 (F(1,27) = 4.24, p < 0.05) effects on the current necessary to elicit 20 APs in 1 s. Before 10 μm NS309 application, significantly less current was needed in SOM neurons from conditioned mice to elicit 20 APs (pre-NS309 ANOVA: F(2,27) = 5.27, p < 0.05; *Fischer's PLSD p < 0.05). NS309 (10 μm) increased the amount of current required to elicit 20 APs in neurons from all training groups (#paired t tests p < 0.05). After NS309, there was no difference between groups in the amount of current required to elicit 20 action potentials. n: naive 8, pseudo 11, conditioned 11. Open circle, Predrug; filled circle, postdrug.
Tables
Naive (n = 32) Pseudo (n = 26) Conditioned (n = 33) Vrest (mV) −65.5 ± 0.8 −64.3 ± 0.7 −63.4 ± 0.6 Input resistance (MΩ) 84.2 ± 6.3 85.4 ± 4.9 87.8 ± 7.2 Sag (mV) 3.82 ± 0.43 3.54 ± 0.26 3.64 ± 0.32 AP threshold (mV) −50.8 ± 1.0 −50.8 ± 1.0 −52.1 ± 0.7 AP height (mV) 89.2 ± 3.8 90.1 ± 3.8 88.1 ± 3.4 AP half width (ms) 1.37 ± 0.07 1.22 ± 0.08 1.35 ± 0.08 Time to spike (ms) 7.83 ± 0.49 6.91 ± 1.74 7.33 ± 0.49 Vrest is the resting membrane potential immediately after breaking into the cell. Input resistance is calculated as the slope of the IV curve. Sag is the difference between the peak and steady state hyperpolarization in response to an 800 ms current injection. Action potentials were elicited by activation of the Schaffer collaterals. Threshold was measured where the first derivative of the upslope of the trace equals 20 mV/ms. Height is the difference between the baseline and maximal depolarization. Half width is width of the action potential at half of the height. Time to spike is the time from the onset of the stimulus artifact to the peak of the action potential. n, Number of neurons.
Naive (n = 10) Pseudo (n = 9) Conditioned (n = 10) Input/output 4.42 ± 0.52 4.71 ± 0.38 4.68 ± 0.49 EPSP rise slope 0.38 ± 0.03 0.38 ± 0.04 0.39 ± 0.03 EPSP summation (mV*s) 8.55 ± 1.01 10.64 ± 1.26 9.82 ± 1.02 EPSP properties were recorded in the presence of GABA blockers. Input/output was measured as the slope of the line of the normalized stimulation intensity and the amplitude of the EPSP. EPSP rise slope is the rise slope of the largest subthreshold EPSP. EPSP summation is measured as the area of five subthreshold EPSPs delivered at 50 Hz from stimulation initiation to return to baseline membrane potential. None of these measures were significantly altered by learning. n, Number of neurons.
- Table 3.
Properties of spontaneous and miniature IPSCs measured from CA1 pyramidal neurons
sIPSCs mIPSCs Naive (n = 20) Pseudo (n = 27) Conditioned (n = 22) Naive (n = 18) Pseudo (n = 19) Conditioned (n = 19) Amplitude (pA) 18.25 ± 1.37 21.17 ± 0.97 20.48 ± 1.53 10.45 ± 0.29 10.67 ± 0.36 11.94 ± 0.82 Area (pA*ms) 174.1 ± 14.8 222.2 ± 20.6 197.4 ± 15.1 81.98 ± 7.76 82.54 ± 6.88 99.73 ± 10.43 Rise 3.53 ± 0.16 3.87 ± 0.15 4.15 ± 0.18 1.97 ± 0.06 1.89 ± 0.08 2.12 ± 0.09 Decay 5.84 ± 0.29 6.27 ± 0.33 5.83 ± 0.30 3.23 ± 0.16 2.88 ± 0.17 3.33 ± 0.24 Spontaneous and miniature IPSCs were recorded onto CA1 pyramidal neurons. mIPSCs were recorded in the presence of 500 nM TTX. While there was a significant difference in the number of both IPSCs and EPSC (see Fig. 3), there was no significant difference in the amplitude, area, rise, or decay. n, Number of neurons.
Naive (n = 25) Pseudo (n = 32) Conditioned (n = 32) Vrest (mV) −49.82 ± 1.87 −49.20 ± 1.65 −50.57 ± 1.26 Input resistance (MΩ) 249.3 ± 42.6 224.1 ± 35.2 260.7 ± 51.8 AP threshold (mV) −44.08 ± 2.33 −41.29 ± 1.15 −43.14 ± 1.52 AP height (mV) 70.04 ± 3.18 71.10 ± 5.32 70.96 ± 3.29 AP half width (ms) 1.27 ± 0.11 1.39 ± 0.10 1.30 ± 0.13 Sag (mV) 2.79 ± 0.16 2.64 ± 0.22 2.57 ± 0.19 Vrest is the resting membrane potential immediately after breaking into the cell. Input resistance is calculated as the slope of the IV curve. Action potentials were elicited by current injection through the recording electrode. Threshold was measured where the first derivative of the upslope of the trace equals 20 mV/ms. Height is the difference between the baseline and maximal depolarization. Half width is the width of the action potential at half of the height. Sag was measured as the difference in the peak versus the steady state hyperpolarization to a −100 pA current injection from a holding potential of −60 mV. n, Number of neurons.
