Neuroadaptation of GABAergic transmission in the central amygdala during chronic morphine treatment

Abstract

We investigated possible alterations of pharmacologically-isolated, evoked GABA(A) inhibitory postsynaptic potentials (eIPSPs) and miniature GABA(A) inhibitory postsynaptic currents (mIPSCs) in the rat central amygdala (CeA) elicited by acute application of µ-opioid receptor (MOR) agonists (DAMGO and morphine; 1 µM) and by chronic morphine treatment with morphine pellets. The acute activation of MORs decreased the amplitudes of eIPSPs, increased paired-pulse facilitation (PPF) of eIPSPs and decreased the frequency (but not the amplitude) of mIPSCs in a majority of CeA neurons, suggesting that acute MOR-dependent modulation of this GABAergic transmission is mediated predominantly via presynaptic inhibition of GABA release. We observed no significant changes in the membrane properties, eIPSPs, PPF or mIPSCs of CeA neurons during chronic morphine treatment compared to CeA of naïve or sham rats. Superfusion of the MOR antagonist CTOP (1 µM) increased the mean amplitude of eIPSPs in a majority of CeA neurons to the same degree in both naïve/sham and morphine-treated rats, suggesting a tonic activation of MORs in both conditions. Superfusion of DAMGO decreased eIPSP amplitudes and the frequency of mIPSCs equally in both naïve/sham and morphine-treated rats but decreased the amplitude of mIPSCs only in morphine treated rats, an apparent postsynaptic action. Our combined findings suggest the development of tolerance of the CeA GABAergic system to inhibitory effects of acute activation of MORs on presynaptic GABA release and possible alteration of MOR-dependent postsynaptic mechanisms that may represent important neuroadaptations of the GABAergic and MOR systems during chronic morphine treatment.

Figure 1. Acute application of both DAMGO and morphine decrease GABA_A-IPSPs in most CeA neurons

We applied 1 μM DAMGO and 1 μM morphine acutely by rapid superfusion and carried out intracellular recordings using sharp electrodes in current-clamp mode from CeA neurons held close to their RMPs. A) and B): normalized input-output curves showing a decrease in the amplitude of eIPSPs elicited by DAMGO and morphine, at all and most of the stimulation intensities, respectively. C) DAMGO and D) morphine also decreased the mean amplitudes of eIPSPs evoked by the ½ maximal IPSP protocol, consisting of 4 repetitive stimuli at a half-maximal intensity. The upper half of the panels shows representative IPSP traces. E) and F): Paired-pulse ratios (mean amplitudes of IPSP2/IPSP1) used to estimate PPF suggest that DAMGO and morphine effects on eIPSPs are predominantly mediated by inhibition of presynaptic GABA release. The upper half of the panels shows representative traces from the PPF protocol using 50 ms inter-stimulus intervals. All results expressed as mean ± s.e.m., and statistical significance (* P < 0.05) calculated by one-sample t-test/Wilcoxon signed rank test.

Figure 2. Acutely applied morphine decreases frequency but not amplitude of miniature GABA_A-IPSCs

A) Representative traces of whole-cell recordings of spontaneous mIPSCs from CeA neurons, with 1 μM TTX in the bath to block action potential-dependent events. B) Morphine (1 μM) applied acutely shifted the cumulative frequency of mIPSCs to the right (suggesting a decrease), but had no effect on the amplitude of the mIPSCs as shown in a representative cell (C). The results represent cumulative probability determined by the Kolmogorov-Smirnov test in a representative CeA neuron. D) The averaged mIPSC frequencies recorded from multiple CeA neurons were decreased by acute application of morphine, whereas the averaged amplitudes of mIPSCs were unchanged (E). The results are expressed as mean ± s.e.m., and statistical significance (*) calculated by one-sample t-test/Wilcoxon signed rank test was set at P < 0.05.

Figure 3. No difference in basal CeA IPSPs between naïve/sham and chronic morphine-treated rats

We implanted 2 placebo or morphine pellets s.c. and performed intracellular recording on day 6 after the pellet implantation. In CeA slices from chronic morphine-treated rats, we added 1 μM morphine to the bath to avoid morphine withdrawal. A) Neither input-output curves nor B) the mean ½ max IPSP protocol showed significant alteration of the amplitude of eIPSPs during chronic morphine treatment. C) Basal eIPSP PPF in CeA neurons from chronic morphine rats also was not different from that of naïve/sham rats. Results expressed as mean ± s.e.m and statistical significance P < 0.05 determined by Student’s t-test.

Figure 4. Antagonism of MORs increases IPSP amplitudes in CeA neurons from both naïve/sham and chronic morphine-treated rats

MOR antagonist CTOP (1 μM) superfused acutely during intracellular recordings with sharp electrodes. A) There was significant alteration of input-output curves by CTOP represented by an increase in the mean eIPSP amplitudes at the 2 and 1 intensity (normalized) stimuli in naïve/sham and chronic morphine rats, respectively. B) CTOP (1 μM) significantly increased the mean amplitude of eIPSPs determined by the ½ maximal IPSP protocol in CeA of both animal groups, with little recovery after washout of CTOP. C) CTOP increased eIPSP PPF in approximately half the CeA neurons (right bars) and decreased PPF in the other half. All the results expressed as mean ± s.e.m. and statistical significance * P < 0.05 calculated by one-sample t-test/Wilcoxon signed rank test.

Figure 5. MOR-dependent decrease in IPSP amplitudes in CeA neurons persists in chronic morphine-treated rats compared to naïve/sham rats

MOR agonist DAMGO (1 μM) superfused acutely during intracellular recordings with sharp electrodes. In the morphine treated rats, morphine sulphate (1 μM) was present in the bath during recording to prevent morphine withdrawal A) DAMGO (1 μM) significantly decreased (**) the mean amplitude of eIPSPs determined by the ½ maximal IPSP protocol in CeA of both animal groups. B) DAMGO increased PPF in most of the CeA neurons from both animal groups. All the results are expressed as mean ± s.e.m. and statistical significance *, # P < 0.05 and ** P < 0.01 were calculated by one-sample t-test/Wilcoxon signed rank test.

Figure 6. Antagonism of MORs increases the frequency of miniature IPSCs: whole-cell recordings with 1 μM TTX in the bath and 1 μM CTOP superfused acutely

A) and F): representative recordings of the mIPSPs. In a representative CeA neuron of naïve/sham rats, CTOP significantly shifted frequency to the left (frequency increase) (B), with no significant change in cumulative amplitude (C). The graphs represent cumulative probability determined by the Kolmogorov-Smirnov test. The averaged data from multiple cells also showed increased frequencies of mIPSCs (D) elicited by acute application of CTOP and unchanged mIPSC amplitudes (E). Results are expressed as mean ± s.e.m. and statistical significance (*) P < 0.05 was calculated by one-sample t-test/Wilcoxon signed rank test. G) During chronic morphine treatment, CTOP shifted the cumulative probability of frequency to the left with little change in the amplitude (H), as recorded in a representative CeA neuron. Acute application of CTOP increased the averaged mIPSCs frequency (I) determined in multiple CeA neurons, but had no significant effects on the averaged amplitudes of mIPSCs (J).

Figure 7. Activation of MORs decreases only the frequency of CeA mIPSCs in naive/sham rats but decreases both the frequency and amplitude of mIPSCs in morphine-treated rats: whole-cell recordings with 1 μM TTX in the bath and 1 μM DAMGO superfused acutely

A) and F): representative recordings of the mIPSPs. B) and C) represent cumulative probability determined by the Kolmogorov-Smirnov test in a representative CeA neuron from a naïve/sham rat. DAMGO significantly shifted inter-event intervals to the right (frequency decrease), with no significant change in cumulative amplitude. D) The averaged data from multiple cells also showed decreased frequencies of mIPSCs elicited by acute application of DAMGO, but unchanged mIPSC amplitudes (E). G) During chronic morphine treatment, DAMGO shifted the cumulative probability of mIPSC intervals to the right (frequency increase) and of amplitude to the left (amplitude decrease) (H), as recorded in a representative CeA neuron. Acute application of DAMGO decreased both the mean mIPSC frequency (I) as well mean amplitude of mIPSCs (J), as determined in multiple CeA neurons. Results are expressed as mean ± s.e.m. and statistical significance (*) P < 0.05 and (**) P < 0.01 were calculated by one-sample t-test/Wilcoxon signed rank test. n = 5 CeA neurons from naive/sham rats; n = 7 from chronic morphine rats.