Article Figures & Data
Figures
- Figure 1.
Location of stimulating and recording electrodes in the amygdala slice preparation and experimental time line. A, Diagram of placement of stimulating (S) and recording electrodes (R) electrodes in a coronal section of rat brain modified from the atlas of Paxinos and Watson (1998). IC, Internal capsule. B, Time line for experimental fear conditioning paradigm and in vitro experiments.
- Figure 2.
NMDA-mediated synaptic transmission is not altered in lateral amygdala neurons, and PPF of NMDA EPSCs is reduced in fear conditioning. A, NMDA EPSCs recorded in Mg2+-free ACSF and 5 μm NBQX are not changed in fear-conditioned animals. NMDA EPSC amplitudes are plotted as a function of stimulus intensity in neurons from both control (naive, filled circles; unpaired, filled triangles) and fear-conditioned (open circles) rats. The slopes of the lines were not significantly different (p > 0.05) in neurons from the three populations of animals. Insets show superimposed traces of EPSCs in neurons from the different animal populations. B, The relationship between EPSC amplitude and stimulus intensity in control ACSF in the presence and absence of d-APV (25 μm) shows that the effect of APV is reduced in FC (right) compared with naive control (NC; left) and unpaired control (UP; middle) animals. Note that scales in the control groups are similar but are smaller in the FC group because of the large size of AMPA EPSCs in that group. C, PPF of the NMDA EPSC is decreased in lateral amygdala neurons from fear-conditioned rats. Percentage of facilitation is plotted as a function of interstimulus interval (in milliseconds). The horizontal dashed line is the 0 reference line. PPF is significantly decreased (p < 0.0001) in neurons from fear-conditioned animals compared with control (naive and unpaired) rats. Insets show traces of NMDA receptor-mediated PPF in lateral amygdala neurons from the three populations of animals at the same interstimulus interval (35 msec). VH = -60 mV.
- Figure 3.
Concentration-response relationship for NMDA-induced currents is shifted to the right in projection neurons in the lateral amygdala of fear-conditioned animals (filled circles), compared with naive (open circles) and unpaired (filled triangles) control animals. Responses are elicited by drop-applying different concentrations of NMDA to the input of the recording chamber in Mg2+-free ACSF in the presence of 5 μm NBQX (to block AMPA receptors) and 1 μm TTX (to block action potential-dependent neurotransmitter release) (VH = -64 mV). A washout period of 15 min (for low concentrations) to 35 min (for high concentrations) is allowed between applications. NMDA-induced current in unpaired control rats is not significantly different from naive rats but is significantly different from fear-conditioned animals. There is also a significant difference in NMDA-induced current recorded in neurons from naive control and fear-conditioned rats. Data are expressed as mean ± SEM NMDA current. Numbers in parentheses are the number of data points for each mean value.
- Figure 4.
Inhibitory effect of ifenprodil on synaptic NMDA receptors is reduced in neurons from fear-conditioned animals. Ifenprodil (10 μm), an antagonist of the NR2B subunit, reduced NMDA EPSCs recorded in amygdala neurons from unpaired control rats but had a diminished effect in neurons from fear-conditioned animals. The NMDA component of the EPSC is isolated using the AMPA receptor antagonist CNQX in Mg2+-free ACSF. Stimulus intensity, 9 V.
- Figure 5.
Expression of NR2A and NR2B proteins is reduced in fear conditioning. NR2A and NR2B subunits are identified at 180 kDa in a representative Western blot of rat amygdala tissue. In FC rats, the NR2A and NR2B expression is significantly reduced (A or C) compared with that in amygdalas from UP and naive control (NC) animals. Bar graphs summarizing the effects of fear conditioning on NR2A and NR2B subunit expression are shown in B and D, respectively. The expression of NR2A and NR2B protein is significantly reduced (*p < 0.05) in fear-conditioned rats (NR2A, n = 10; NR2B, n = 12) when compared with that from naive (NR2A, n = 7; NR2B, n = 8) and unpaired (NR2A, n = 8; NR2B, n = 6) rats. MW, Molecular weight.
- Figure 6.
Expression of phosphorylated-NR1 (PNR1) but not NR1 subunit protein is decreased in fear conditioning. A, Representative Western blots for NR1 subunits having a molecular weight (MW) of ∼130 kDa show that NR1 expression is not significantly altered in amygdalas from fear-conditioned rats. B, Bar graphs show that the NR1 subunit is not changed in amygdalas from FC animals (n = 8; p > 0.05) compared with those from naive control (NC; n = 8) and UP (n = 8) rats. The NR1 proteins from unpaired and fear-conditioned rats are shown as a percentage of those from naive rats. C, Representative Western blots of PNR1 from amygdalas of naive control, unpaired control, and fear-conditioned animals. PNR1 subunit expression appears at ∼130 kDa and shows a reduced expression in amygdalas from fear-conditioned animals. D, Bar graphs summarizing the expression of PNR1 protein from the amygdalas of the three populations of animals. PNR1 expression in the unpaired control and fear-conditioned group is expressed as a percentage of the naive control group. Protein expression of PNR1 was significantly decreased in FC animals (n = 6; *p < 0.05) compared with NC (n = 6) and UP (n = 6) animals.
