Figure 1.
5-HT enhances spontaneous excitatory postsynaptic currents (sEPSCs) in mPFC layer V pyramidal neurons. (A) A representative neuron showing that application of 5-HT (30 μM) for 3 min dramatically increases both the frequency and amplitude of sEPSCs. (B) Cumulative interevent interval distribution illustrating a significant decrease in the interevent interval (i.e., increased frequency; p < 0.01,Kolmogorov-Smirnov test) during 5-HT application. (C) Cumulative
distribution of sEPSC amplitude before and after 5-HT application. (D) and (E) The bar graph showing the summary data of the effect of 5-HT on the average amplitude and frequency of sEPSCs. The total number of animals examined is shown in parentheses. Data are presented as mean ± SEM. Asterisk, significant difference from baseline at p < 0.05.
The data shown in A to C were taken from the same neuron of a rat treated saline for 5 days followed by a 3-day withdrawal. Holding potential, -70 mV.
Figure 2.
Repeated cocaine administration decreases 5-HT-induced enhancement of sEPSCs in the mPFC. (A) An example experiment showing the effect of 5-HT (30 μM) on sEPSCs recorded from a mPFC layer V pyramidal neuron of a rat treated with cocaine for 5 days followed by a 3-day withdrawal. (B) Cumulative probability plots depicting the effect of 5-HT on the distribution of sEPSC interevent interval for the experiment illustrated in (A). (C) Cumulative distribution of sEPSC amplitude recorded before and after application of 5-HT for the same experiment. (D) and (E) The bar graph showing the summary data of34
(F) and (G) The bar graph showing the summary data of the effect of 5-HT on the average amplitude and frequency of sEPSCs from rats that were injected daily with saline or cocaine for 5 days and then examined at 1, 3, 7, or 14 days after withdrawal. The total number of animals examined is shown in parentheses. Data are presented as mean ± SEM.
Asterisk, significant difference from pre-5-HT baseline at p < 0.05.
Pound sign, significant difference from saline-treated group at p < 0.05.
Holding potential, -70 mV.
Figure 3.
Activation of 5-HT2A receptors mediates the 5-HT-induced enhancement of sEPSCs in mPFC layer V pyramidal neurons. (A) and (B) The bar graph summarizing the effects of 5-HT (30 μM) on the average amplitude and frequency of sEPSCs in the presence or absence of various 5-HT receptor antagonists. (C) Top, an example experiment showing the effect of DOI (10 μM) on sEPSCs recorded from a mPFC layer V pyramidal neuron of a saline-treated rat. Bottom, the bar graph summarizing the effect of DOI on the average amplitude and frequency of sEPSCs. (D) Top, an example experiment showing the effect of DOI on sEPSCs recorded from a mPFC layer V pyramidal neuron of a rat 3 days after withdrawal from 5 days of cocaine treatment. Bottom, the bar graph summarizing the effect of DOI on the average amplitude and frequency of sEPSCs. The total number of animals examined is shown in parentheses. Data are presented as mean ± SEM. Asterisk, significant difference from pre-DOI baseline at p < 0.05. Holding potential, -70 mV.35
Figure 4. Repeated cocaine administration decreases 5-HT
2A receptor-mediated serotonergic enhancement of sEPSCs via the activation of 5-HT2Areceptors. (A) and (B) Summary of experiments showing the effects of 5-HT (30 μM) on the average amplitude and frequency of sEPSCs in mPFC layer V pyramidal neurons of rats receiving vehicle, SCH23390 (0.5 mg/kg), raclopride (0.5 mg/kg), ketanserin (1 mg/kg), RS102221 (2 mg/kg), or prazosin (1 mg/kg) 30 min before saline or cocaine injection for 5 days followed by a 3-day withdrawal. The total number of animals examined is shown in parentheses. Data are presented as mean
± SEM. Asterisk, significant difference from pre-5-HT baseline at p <
0.05. Pound sign, significant difference from saline-treated group at p
< 0.05. Holding potential, -70 mV.
Figure 5.
Repeated DOI administration decreases 5-HT-induced enhancement of sEPSCs in the mPFC. (A) An example experiment showing the effect of 5-HT (30 μM) on sEPSCs recorded from a mPFC layer V pyramidal neuron of a rat treated DOI (1 mg/kg/day) for 5 days followed by a 3-day withdrawal. (B) Cumulative probability plots depicting the effect of 5-HT on the distribution of sEPSC interevent interval for the experiment illustrated in (A). (C) Cumulative distribution of sEPSC amplitude recorded before and after application of 5-HT for the same experiment.(D) and (E) The bar graph showing the summary data of the effect of 5-HT on the average amplitude and frequency of sEPSCs. (F) and (G) The bar graph showing the summary data of the effect of 5-HT on the average amplitude and frequency of sEPSCs from rats that were injected daily with saline or DOI for 5 days and then examined at 1, 3, or 7 days
36
parentheses. Data are presented as mean ± SEM. Asterisk, significant difference from pre-5-HT baseline at p < 0.05. Pound sign, significant difference from saline-treated group at p < 0.05. Holding potential, -70 mV.
Figure 6.
Repeated cocaine treatment increases phosphorylation of Gαq/11 proteins in the mPFC. (A) Top, representative immunoblot showing the levels of 5-HT2A receptors in the mPFC slices from rats 3 days after withdrawal from 5 days of saline or cocaine treatment. Bottom, group data showing the normalization of 5-HT2A receptors to the β-actin was determined in each group of six separate experiments. (B) Left, the representative immunoblot showing the localization of 5-HT2A receptors in the detergent-resistant membrane (DRM) fractions. Nine fractions were collected from the top and immunoblotted for the indicated proteins.Caveolin-1 was used as DRM marker, whereas transferrin receptor (TfR) was used as the non-DRM marker. Right, quantitative analysis of the 5-HT2A receptor distribution was performed by densitometry and calculated in the percentage of the total protein amount in all fractions.
(C) Top, representative immunoblot showing the levels of phosphorylated Gαq/11 proteins in the mPFC slices from rats that were injected daily with saline or cocaine for 5 days and then examined at 1, 3, or 7 days after withdrawal. Bottom, group data showing the normalization of phosphor-Gαq/11 proteins to the non-phosphorylated form was determined in each group of four experiments. (D) Top, representative immunoblot showing the levels of phosphorylated Gαq/11
37
proteins in the mPFC slices from rats receiving vehicle, SCH23390 (0.5 mg/kg), raclopride (0.5 mg/kg), or ketanserin (0.5 mg/kg) 15 min before saline or cocaine injection for 5 days followed by a 3-day withdrawal.
Bottom, group data showing the normalization of phosphor-Gαq/11
proteins to the non-phosphorylated form was determined in each group of four experiments. (E) Top, representative immunoblot showing the levels of RGS4 in the mPFC slices from rats 3 days after withdrawal from 5 days of saline or cocaine treatment. Bottom, group data showing the normalization of RGS4 to the β-actin was determined in each group of five separate experiments. The total number of animals examined is shown in parentheses. Data are presented as mean ± SEM.
Asterisk, significant difference from saline-treated group at p < 0.05.
Figure 7. Repeated cocaine administration decreases 5-HT-induced inward
currents in the mPFC. (A) Summary of experiments showing the 5-HT (30 μM)-induced inward currents recorded with electrode filled with intracellular solution containing GTP (0.3 mM) in mPFC layer V pyramidal neurons from rats treated with saline ({) or cocaine (z) for 5 days followed by a 3-day withdrawal. (B) Summary of experiments showing the 5-HT (30 μM)-induced inward currents recorded with electrode filled with intracellular solution containing GTPγS (0.3 mM) in mPFC layer V pyramidal neurons from rats treated with saline ({) or cocaine (z) for 5 days followed by a 3-day withdrawal. Data are presented as mean ± SEM. The dashed line represents 0 pA.Interevent interval (ms)
0 200 400 600 800
Cumulati ve probabil it y
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Baseline 5-HT Wash (P < 0.01; K-S test)
0 .1 se c 5 p A
Baseline
5-HT
Figure 1. Huang et al., 2008
C
Amplitude (pA)
0 5 10 15 20 25 30 35
Cumulative p robability
0.0 0.2 0.4 0.6 0.8 1.0 1.2
Baseline 5-HT Wash (P = 0.03; K-S test)