Glutamate-mediated excitatory postsynaptic currents (EPSCs) were recorded under whole-cell voltage-clamp in NRM slices in vitro. The EPSCs were compared between NRM slices from saline-treated control rats and those from morphine-treated tolerant rats. Both groups of slices (control and tolerant) were maintained in 5 μM morphine throughout recording experiment in vitro to prevent morphine withdrawal (Ingram et al., 1998). A separate group of control slices kept in a morphine-free solution (normal group) was used as controls for the acute morphine added. We used the paired-pulse ratio (PPR) to assess chronic morphine-induced changes in glutamate synaptic transmission in the two types of NRM neurons, primary cells and secondary cells. In the μ receptor-containing secondary cells in control slices, the average PPR was 1.76 ± 0.05 (n = 16), indicating a common synaptic facilitation (PPR>1) by the two stimuli. However, in secondary cells from morphine-tolerant rats, the PPR was significantly smaller than that in controls (1.43 ± 0.07, n = 41, P < 0.01, Fig. 1A,C), indicating an increased probability of presynaptic glutamate release in these secondary cells in a morphine-tolerant state. The PPR in secondary cells in the normal group without 5 μM morphine was 1.89 ± 0.13 (n = 33), which was not statistically different from that in control cells kept in 5 μM morphine (p > 0.05), excluding possible effect of the acute morphine on the PPR. In contrast to secondary cells, there was no significant difference between the EPSC PPRs in μ receptor-lacking primary cells from control and from morphine-tolerant rats (control, 1.43 ± 0.11, n = 10; tolerant, 1.44 ± 0.08, n = 17; P > 0.05, Fig 1B,C). These data indicate that chronic morphine increases glutamate synaptic transmission selectively in the μ receptor-containing secondary cells in a morphine-tolerant state.

In control secondary cells, bath application of the adenylyl cyclase (AC) activator forskolin (10 μM) significantly increased the amplitude of evoked EPSCs (eEPSCs) by 53.9 ± 4.1% (control, 95.1 ± 7.3 pA, forskolin, 144.1 ± 14.2 pA, n = 12, P < 0.01). Forskolin produced a comparable EPSC increase in secondary cells in normal slices without 5 μM morphine (57.4 ± 11.4%, n = 6, p < 0.01, p > 0.05 when compared to its effect in control slices with 5 μM morphine). However, in tolerant secondary cells, the EPSC-enhancing effect of forskolin was significantly increased to 95.3 ± 5.3% (p < 0.01, compared to its effect in control group) (control, 88.9 ± 7.8 pA, forskolin, 174.3 ± 13.9 pA, n = 7, P < 0.01, Fig. 2A,B). Similar to the effect of chronic morphine, forskolin (10 μM) also significantly decreased the PPR of glutamate EPSCs in secondary cells from both saline-treated and morphine-tolerant rats (saline: control, 1.73 ± 0.21, forskolin, 1.03 ± 0.14, n = 5, P < 0.01; tolerant: control, 1.43 ± 0.14, forskolin, 0.90 ± 0.03, n = 5, P < 0.01, Fig 2C). Due to a significant decrease in the basal PPR, direct comparison in percentage term of the forskolin effects between the two groups was not feasible. In primary cells, by contrast, although forskolin increased the eEPSC amplitude in both control and tolerant groups, the magnitude of its effects in the two groups was not changed (control, 72.8 ± 17.6%, n = 7, tolerant, 67.0 ± 14.9%, n = 5, P > 0.05).

To further confirm the forskolin effect on presynaptic glutamate release, we examined forskolin action on miniature EPSCs (mEPSCs) in NRM neurons. In control secondary cells, forskolin (10 μM) increased the frequency, but not the amplitude, of glutamate mEPSCs (frequency: control, 5.70 ± 1.03 Hz, forskolin, 8.46 ± 1.07 Hz, n = 5, P < 0.05; amplitude: control, 20.8 ± 2.0 pA, forskolin, 20.6 ± 2.4 pA, n = 5, P > 0.05). This forskolin effect was also observed in secondary cells from tolerant rats (frequency: control, 7.64 ± 0.96 Hz, forskolin, 15.18 ± 0.98 Hz, n = 5, P < 0.01; amplitude: control, 21.3 ± 2.4 pA, forskolin, 20.5 ± 2.23 pA, n = 5, P > 0.05). By comparison, the forskolin-induced increase in the mEPSC frequency was significantly greater in tolerant secondary cells (control, 54.2 ± 9.8%, tolerant, 104.3 ± 11.5%, n = 5, P < 0.05, Fig 3 & Fig. 4D). The forskolin-induced increase in mEPSC frequency in tolerant primary cells was not altered (control, 62.6 ± 17.4%, n = 4, tolerant, 72.2 ± 36.4%, n = 5, P > 0.05).

The cAMP analog 8-bromo-cAMP (8-br-cAMP) produced similar effects to those of forskolin. Bath application of 8-br-cAMP (1 mM) increased the eEPSC amplitude in secondary cells from both saline control and morphine-tolerant rats (saline: control, 110.9 ± 14.8 pA, 8-br-cAMP, 166.6 ± 9.2 pA, n = 4, P < 0.01; tolerant: control, 130.1 ± 1.4 pA, 8-br-cAMP, 260.2 ± 28.5 pA, n = 7, P < 0.001). This cAMP effect was significantly enhanced by chronic morphine treatment (saline, 52.1 ± 9.4%, tolerant, 103.6 ± 10.1%, p < 0.05). 8-br-cAMP also decreased the PPR in secondary cells from both saline control and tolerant rats (saline: control, 1.70 ± 0.08, 8-br-cAMP, 1.10 ± 0.07, n = 4, P < 0.01; tolerant: control, 1.49 ± 0.17, 8-br-cAMP, 1.14 ± 0.10, n = 5, P < 0.05, Fig 4A,B).

Bath application of 8-br-cAMP (1 mM) also significantly increased the frequency of mEPSC in secondary cells from both saline control and tolerant rats (saline: control, 4.72 ± 0.47 Hz, 8-br-cAMP, 6.85 ± 0.70 Hz, n = 8, p < 0.01; tolerant: control, 6.62 ± 0.88 Hz, 8-br-cAMP, 12.46 ± 1.25 Hz, n = 7, p < 0.01, Fig. 4C). However, similar to the forskolin effect, this effect of 8-br-cAMP was also significantly enhanced in the slices from morphine-tolerant rats (saline, 46.9 ± 7.9%, n = 8, tolerant, 97.5 ± 21.2%, n = 7, p < 0.05, Fig. 4D). 8-br-cAMP did not change the amplitudes of mEPSCs in either group (saline: control, 15.9 ± 0.6 pA, 8-br-cAMP, 15.9 ± 1.3 pA, n = 8, P > 0.05; tolerant: control, 18.8 ± 1.2 pA, 8-br-cAMP, 18.9 ± 1.6 pA, n = 7, P > 0.05).

The enhanced forskolin and 8-br-cAMP effects on eEPSC amplitude and mEPSC frequency in morphine-tolerant rats indicate a sensitized or upregulated AC system in a morphine-tolerant state.

To determine the involvement of the AC pathway in the chronic morphine-induced increase of glutamate release in NRM secondary cells, NRM slices were treated with MDL12330a, a selective AC inhibitor. While pre-incubation with MDL12330a (100 μM) did not change the EPSC PPR in secondary cells from control rats (control, 1.71 ± 0.06, MDL, 1.76 ± 0.03, n = 5, p > 0.05), it largely reversed the chronic morphine-induced PPR decrease in secondary cells from morphine-tolerant rats (tolerant, 1.43 ± 0.07, +MDL, 1.74 ± 0.07, n = 16, P < 0.01, Fig. 5A,B,C). Bath application of MDL12330a (100 μM) also significantly decreased the frequency of mEPSCs in tolerant secondary cells (tolerant, 10.17 ± 0.89 Hz, +MDL, 5.86 ± 0.66 Hz, n = 5, P < 0.01, Fig. 5D,E,F) whereas it did not alter the mEPSC amplitude (tolerant, 16.9 ± 1.1 pA, +MDL, 18.1 ± 1.2 pA, n = 5, P > 0.05). MDL had no effect on either the frequency or the amplitude of mEPSCs in secondary cells from saline control rats (frequency: control, 4.39 ± 0.45 Hz, +MDL, 4.71 ± 0.50 Hz, n = 5, P > 0.05; amplitude: control, 16.9 ± 2.0 pA, +MDL, 17.5 ± 1.5 pA, n = 5, P > 0.05, Fig. 5F). Next, we used a PKA inhibitor to determine whether PKA was involved in the enhanced glutamate transmission in secondary cells induced by chronic morphine. Pre-incubation with H89 (10 μM) largely inhibited the chronic morphine-induced decrease of the EPSC PPR in secondary cells from morphine-tolerant rats (tolerant, 1.43 ± 0.07, +H89, 1.71 ± 0.06, n = 13, P < 0.01, Fig. 6A,B). H89 was without effect on the EPSC PPR in secondary cells from control rats (tolerant, 1.76 ± 0.06, +H89, 1.90 ± 0.08, n = 6, P > 0.05, Fig. 6B). Similarly, bath application of H89 (10 μM) significantly decreased the frequencies, but not the amplitude, of mEPSCs in tolerant secondary cells (frequency: tolerant, 8.55 ± 2.42 Hz, +H89, 4.66 ± 2.11 Hz, n = 5, P < 0.05; amplitude: tolerant, 15.1 ± 0.9 pA, +H89, 14.3 ± 0.7 pA, n = 5, P > 0.05, Fig 6C,D). In control secondary cells, H89 did not change either the frequency or the amplitude of mEPSCs (frequency: control, 3.96 ± 0.44 Hz, +H89, 3.43 ± 0.62 Hz, n = 5, P > 0.05; amplitude: control, 20.5 ± 3.8 pA, +H89, 20.2 ± 3.0 pA, n = 5, P > 0.05, Fig. 6D).

These data obtained with both MDL12330a and H89 suggest that the cAMP/PKA pathway is critically involved in the chronic morphine-induced enhancement of glutamate synaptic transmission in NRM secondary cells.

Finally, we examined whether the PKC pathway was also involved in the chronic morphine effect on glutamate EPSCs. Phorbol 12-myristate 13-acetate (PMA, 1 μM), a phorbol ester activator of PKC, increased the amplitude of eEPSCs in NRM secondary cells from both saline control and morphine-tolerant rats (saline: control, 109.9 ± 15.8 pA, PMA, 180.4 ± 22.2 pA, n = 7, P < 0.001; tolerant: control, 134.8 ± 12.9 pA, PMA, 284.7 ± 25.8 pA, n = 7, P < 0.01, Fig. 7A). However, when the PMA effects were compared between the two groups, the EPSC-enhancing effect of PMA was significantly larger in cells from tolerant rats than that in control cells (control, 67.7 ± 6.9%, n = 7, tolerant, 126.6 ± 16.4%, n = 7, p < 0.01, Fig. 7B), indicating a likely upregulated PKC pathway. GF109203X (2 μM), a selective PKC inhibitor, had no significant effect on the EPSC PPR in control secondary cells (control, 1.76 ± 0.14, GF109203X, 1.76 ± 0.05, n = 6, P > 0.05), but it reversed the chronic morphine-induced decrease of the EPSC PPR in secondary cells from morphine-tolerant rats (tolerant, 1.43 ± 0.07, +GF109203X, 1.79 ± 0.10, n = 8, P < 0.05, Fig. 7C,D,E).

In the present study, the effect of chronic morphine on glutamate synaptic transmission was primarily assessed by using the paradigm of EPSC PPR in NRM neurons kept in a tolerant state from morphine-tolerant rats 14. The PPR has been widely used to determine the involvement of a presynaptic site in the mechanism of neurotransmitter release 15161718192021. A change in the PPR is inversely related to the probability of transmitter release and thus, a manipulation that increases the probability of transmitter release results in a reduction in the PPR and vise versa. Since the PPR in a cell under a certain condition is relatively stable, the advantage of using the PPR is to avoid large variance usually present in eEPSC amplitudes and mEPSC frequencies among individual cells, making it possible to compare EPSCs in two separate groups of slices, such as those from saline-treated control rats and those from morphine-tolerant rats. Our current results obtained with the PPR analysis suggest that glutamate synaptic transmission is enhanced through a presynaptic mechanism in NRM secondary cells from morphine-tolerant rats. This conclusion is further supported by the effects of activators and inhibitors of the cAMP/PKA or PKC pathway, which mimicked and reversed the effect of chronic morphine, respectively. It is interesting to note that the chronic morphine-induced EPSC enhancement occurred exclusively in μ receptor-containing secondary cells. This may implicate a functional activation of these μ receptor-expressing NRM cells by glutamate inputs during morphine tolerance (see below). The source of these glutamate inputs is currently unclear.

Presynaptic glutamate release is modulated by many complex and interacting proteins and processes, including components of the cAMP/PKA pathway, in presynaptic terminals 2223. It has been demonstrated that in vitro application of activators of the cAMP/PKA pathway enhances presynaptic release of glutamate in many types of central neurons 2324252627. In vivo administration of chronic morphine induces adaptive hyperactivation of the cAMP/PKA signaling pathway in several brain regions 12728. The hyperactivated cAMP/PKA pathway induced by chronic morphine may induce a series of cellular adaptations, including enhanced transmitter release. However, cAMP-dependent synaptic adaptation induced by in vivo administration of chronic morphine has been reported only in GABAergic synapses in central neurons during morphine withdrawal 212829. The current study provides direct evidence that chronic morphine enhances glutamate synaptic transmission also in a cAMP-dependent way in NRM neurons from morphine-tolerant rats.

Several observations in the current study support a critical role of an upregulated cAMP/PKA pathway in the enhanced glutamate neurotransmission in secondary cells from morphine-tolerant rats. First, the AC activator and cAMP analog mimic the effect of chronic morphine on EPSC PPR. Second, the potency of cAMP activators in enhancing eEPSC amplitude and mEPSC frequency is significantly augmented in neurons from morphine-tolerant rats. Such an augmented effect has been interpreted as the result of a sensitized or upregulated cAMP pathway 212729. Finally, inhibitors of both AC and PKA reverse the chronic morphine-induced EPSC enhancement. This effect of AC/PKA inhibitors observed in tolerant cells, but not in control cells, indicates an elevated basal activity of the cAMP/PKA pathway induced by chronic morphine. Such an upregulation of basal and stimulated PKA activity has been shown in other brain areas 130.

It has been shown that activation of PKC either by the intracellular messenger diacylglycerol or by phorbol esters produces an enhancement of neurotransmitter release including glutamate release in central neurons 233132. Activated PKC may phosporylate a number of proteins in nerve terminal and increase neurotransmitter release by calcium influx through voltage-gated calcium channels or by direct effects on exocytosis or on vesicle-recycling pathways 33. Chronic morphine increases PKC activity in the rat brain and spinal cord 3435. The increased PKC activity, which may be mediated through opioid activation of the phospholipase C pathway coupled to opioid receptors, can phosphorylate AC and increase its adaptive responses to chronic opioids 2730. In the present study, the PKC activator, similar to activators of the cAMP/PKA pathway, exhibited an augmented potency in enhancing glutamate EPSCs in NRM neurons from morphine-tolerant rats, and the PKC inhibitor reversed the effect of chronic morphine. Although non-specific effects of these PKA and PKC inhibitors cannot be completely ruled out, these data indicate a likely upregulated PKC and PKA activity, which is at least partially responsible for the enhanced glutamate synaptic transmission in NRM secondary cells from morphine-tolerant rats.

Extensive evidence has shown that some NRM cells inhibited by μ opioids have a facilitating action on spinal pain transmission through their descending projections 710. Recent pain research shows that those brainstem cells expressing μ-opioid receptors are commonly activated in various chronic pain conditions, including chronic opioid-induced abnormal pain, and contribute to the sensitized pain or hyperalgesia observed during these conditions 11121336. It remains unclear what mediates the activation of these μ receptor-containing cells in those pain conditions. Data from the current study indicate that excessive activity of glutamate synaptic inputs after chronic exposure to opioids may contribute to the activation of these cells during morphine tolerance. An overall effect of this enhanced glutamate synaptic activity in driving these cells needs to be evaluated by taking into account other synaptic inputs such as GABA synaptic transmission.

It has been proposed that the chronic opioid-induced activation of those presumably pain-facilitating cells and consequently, abnormal pain, constitute part of the mechanisms underlying opioid tolerance characterized by a reduced analgesic effect of opioids 36. For example, microinjection of lidocaine into the NRM area to inactivate cell activity reverses the tactile allodynia and thermal hyperalgesia induced by prolonged exposure to morphine, and attenuates morphine tolerance 3738. A critical role of glutamate receptors in the development of opioid tolerance and dependence has been established 3456. The present study illustrates an example of the mechanisms by which glutamate receptors and synapses participate in the development of opioid tolerance. An important role of PKC in opioid tolerance has also been demonstrated in previous studies. For instance, PKC inhibition reduces opioid tolerance 34353940, and PKCγ mutant mice display alleviated morphine tolerance 41.

In summary, the current study shows that chronic morphine enhances glutamate synaptic transmission in μ receptor-containing NRM neurons. This synaptic adaptation appears to be mediated by an upregulated cAMP/PKA and PKC pathway in morphine-tolerant rats. These results may provide key information for pain therapies aiming at inhibiting those brainstem neurons and their descending pain facilitation, which is responsible for the pain sensitization in several chronic pain conditions.