Chronic morphine reduces the readily releasable pool of GABA, a presynaptic mechanism of opioid tolerance

Abstract

Key points: Chronic treatment with opioids, such as morphine, leads to analgesic tolerance. While postsynaptic opioid tolerance is well documented, the involvement of presynaptic mechanisms remains unclear. We show that chronic morphine reduces the ability of periaqueductal grey (PAG) neurons to maintain GABAergic transmission. This depression of GABAergic transmission was due to a reduction in the effective size of the readily releasable pool. This also led to a reduction in opioid presynaptic inhibition; these presynaptic adaptations need to be considered in the development of strategies to reduce opioid tolerance.

Abstract: The midbrain periaqueductal grey (PAG) plays a critical role in tolerance to the analgesic actions of opioids such as morphine. While numerous studies have identified the postsynaptic adaptations induced by chronic morphine treatment in this and other brain regions, the presence of presynaptic adaptations remains uncertain. We examined GABAergic synaptic transmission within rat PAG brain slices from animals which underwent a low dose morphine treatment protocol which produces tolerance, but not withdrawal. Evoked GABAergic IPSCs (inhibitory postsynaptic currents) were less in morphine compared to control saline treated animals. Postsynaptic GABA_A receptor mediated currents and desensitization, presynaptic release probability (P_r ), and inhibition by endogenous neurotransmitters were similar in morphine and saline treated animals. By contrast, the effective size of the readily releasable pool (RRP) was smaller in morphine treated animals. While the μ-opioid agonist DAMGO produced a reduction in P_r and RRP size in saline treated animals, it only reduced P_r in morphine treated animals. Consequently, DAMGO-induced inhibition of evoked IPSCs during short burst stimulation was less in morphine, compared to saline treated animals. These results indicate that low dose chronic morphine treatment reduces presynaptic μ-opioid inhibition by reducing the size of the pool of vesicles available for action potential dependent release. This novel presynaptic adaptation may provide important insights into the development of efficacious pain therapies that can circumvent the development of opioid tolerance.

Keywords: opioid; synaptic transmission; tolerance.

Figure 1. Chronic morphine treatment reduces evoked IPSCs

A, representative averaged traces of paired evoked IPSCs (inter‐stimulus interval 70 ms) at increasing stimulus intensities (5, 20, 50, 90 V) in PAG neurons from animals which had undergone saline, or morphine treatment. The inset shows the ventrolateral subdivision of the caudal‐intermediate PAG where neurons were located. B, summary plot of the stimulus–response relationship for evoked IPSC amplitude and paired pulse ratio in PAG neurons from saline and morphine treated animals (n = 12, 11). C, summary plot of the paired pulse ratio of evoked IPSCs for neurons from saline and morphine treated animals (n = 26, 20) at a mid‐range stimulus intensity (50 V). D, representative traces of evoked IPSCs in neurons from saline and morphine treated animals at the mid‐range stimulus intensity (25 consecutive traces with overlaid average). E, summary plot of the coefficient of variation (CV) of evoked IPSCs for neurons from saline and morphine treated animals at the mid‐range stimulus intensity (averaged response = thick line, n = 26, 22). In B, ^* P < 0.05, ^** P < 0.01 for saline versus morphine. In C and E, box and whiskers plots depict the 25th–75th percentile, and the min–max values, respectively. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2. Chronic morphine treatment does not alter the response to exogenous muscimol and GABA

Raw current traces during application of muscimol (0.1, 0.3 and 1 μm) to isolated PAG neurons from saline (A) and morphine treated (B) animals (n = 6–9). C, concentration–response curves of the steady state current produced by muscimol in isolated PAG neurons and by GABA in neurons in intact PAG slices (concentration in μm). D, the extent of desensitization during application of muscimol (1 μm) to isolated PAG neurons and of GABA (1000 μm) to neurons within intact PAG slices; shown as a percentage of the initial peak current. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3. Chronic morphine treatment does not alter quantal IPSCs

A, representative raw traces of spontaneous miniature IPSCs in neurons from saline and morphine treated animals (n = 17, 15). B, averaged miniature IPSCs in neurons from neurons represented in A (average of 224 and 281 IPSCs over 100 s in saline and morphine neurons). Summary plots of the rate (C), amplitude (D), rise‐time (E) and half‐width (F) of spontaneous miniature IPSCs in neurons from saline and morphine treated animals. In C–E, box and whiskers plots depict the 25th–75th percentile, and the min–max values, respectively. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4. Chronic morphine treatment reduces train evoked IPSCs

Raw traces of IPSCs evoked during repetitive stimulation in PAG neurons from saline (A and B) and morphine treated (C and D) animals (n = 8 per treatment group). Data are shown for trains of 20 stimuli evoked at 20 s⁻¹ (A and C) and 100 s⁻¹ (B and D). Summary plots of phasic charge transfer of evoked IPSCs (E) and tonic charge transfer (F) in saline and morphine treated animals at both stimulus frequencies. The inset in B depicts the calculation of the phasic (i) and tonic (ii) components of charge transfer. In E and F data are also shown for slices from morphine treated animals which were incubated in a cocktail of GPRC antagonists (Antags; see text, n = 7). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5. Chronic morphine treatment reduces the readily releasable pool size

A and B, plots of the phasic charge transfer of evoked IPSCs during train stimulation (100 s⁻¹) in neurons from saline (A) and morphine treated (B) animals (n = 9 each treatment group). The data were fitted by the sum (continuous line) of an exponential decay (dashed line) and recovery (dotted line). C and D, plots of cumulative phasic charge transfer of evoked IPSCs in neurons from saline (C) and morphine treated (D) animals (error bars not included for clarity). RRP size estimates are shown by linear back‐extrapolation from the last 5 IPSCs in the train (RRP_SMN continuous lines, RRP_TR dashed line). The dotted lines are RRP_SMN back‐extrapolations from the 5 IPSCs before the 20th, 30th and 40th evoked IPSCs. E–G, summary plots of the phasic charge transfer of the first evoked IPSC in the train (E), RRP size (F) and probability of release, P _r (G). In G–I, ^* P < 0.05, ^** P < 0.001 for saline versus morphine. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6. The rate of recovery following train stimulation is unaffected by chronic morphine treatment

The charge transfer of single evoked IPSCs at a range of time points following train stimulation. Data are averaged across all neurons from saline and morphine treated animals (n = 6, 7). Data are also shown for the first evoked IPSC in the train. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7. Chronic morphine reduces the DAMGO induced reduction in RRP size

A and B, plots of the phasic charge transfer of evoked IPSCs during train stimulation (50 at 100 s⁻¹) in neurons from saline (A) and morphine treated (B) animals, prior to (Pre) and then during superfusion of DAMGO (n = 7 per treatment group); data were fitted by an exponential decay. C and D, plots of cumulative phasic evoked IPSC charge in PAG neurons from saline (C) and morphine treated (D) animals, prior to and then during DAMGO; RRP_SMN size estimates are shown by linear extrapolation from the last 10 IPSCs in the train. E–G, are charts of the effect of DAMGO on phasic charge transfer of the first evoked IPSC in a train (E), the pool size, RRP_SMN (F), and release probability, P_{r ‐SMN} (G), in PAG neurons from saline and morphine treated animals. ^* P < 0.05, ^** P <  0.01 and ^*** P < 0.0001 for pre‐ versus + DAMGO in each treatment group. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8. Simulation of the effect of reducing P _r and RRP size on evoked IPSCs

Simulated plots of cumulative evoked IPSC amplitude (A) and evoked IPSC amplitude raw traces (B). Data are shown before and after a reduction in both P _r and RRP size (35% and 25% reduction), and after a reduction in P _r alone (35% reduction). Baseline data were normalized, with P _r of 0.1 and replenishment rate, R, of 0.03 (estimated by fitting to the baseline data in Fig. 7 C and D). [Color figure can be viewed at wileyonlinelibrary.com]

Figure 9. Chronic morphine treatment reduces the DAMGO induced inhibition of total charge transfer during repetitive stimulation

Representative raw traces of IPSCs evoked during repetitive stimulation (20 at 100 s⁻¹) in PAG neurons from saline (A) and morphine treated (B) animals prior to and then during superfusion of DAMGO (n = 11, 12 neurons for saline and morphine). C, plot of the inhibition of total charge transfer by DAMGO throughout a train, in PAG neurons from saline and morphine treated animals; P < 0.01–0.05 for saline versus morphine at evoked IPSC3–17 and 19–20. [Color figure can be viewed at wileyonlinelibrary.com]