Opioid tolerance in periaqueductal gray neurons isolated from mice chronically treated with morphine

Abstract

The midbrain periaqueductal gray (PAG) is a major site of opioid analgesic action, and a significant site of cellular adaptations to chronic morphine treatment (CMT). We examined mu-opioid receptor (MOP) regulation of voltage-gated calcium channel currents (I(Ca)) and G-protein-activated K channel currents (GIRK) in PAG neurons from CMT mice. Mice were injected s.c. with 300 mg kg(-1) of morphine base in a slow release emulsion three times over 5 days, or with emulsion alone (vehicles). This protocol produced significant tolerance to the antinociceptive effects of morphine in a test of thermal nociception. Voltage clamp recordings were made of I(Ca) in acutely isolated PAG neurons and GIRK in PAG slices. The MOP agonist DAMGO (Tyr-D-Ala-Gly-N-Me-Phe-Gly-ol enkephalin) inhibited I(Ca) in neurons from CMT mice (230 nM) with a similar potency to vehicle (150 nM), but with a reduced maximal effectiveness (37% inhibition in vehicle neurons, 27% in CMT neurons). Inhibition of I(Ca) by the GABA(B) agonist baclofen was not altered by CMT. Met-enkephalin-activated GIRK currents recorded in PAG slices were significantly smaller in neurons from CMT mice than vehicles, while GIRK currents activated by baclofen were unaltered. These data demonstrate that CMT-induced antinociceptive tolerance is accompanied by homologous reduction in the effectiveness of MOP agonists to inhibit I(Ca) and activate GIRK. Thus, a reduction in MOP number and/or functional coupling to G proteins accompanies the characteristic cellular adaptations to CMT previously described in PAG neurons.

Figure 1

Morphine antinociception is reduced in CMT mice. Mice were treated with morphine base or vehicle for 5 days, as described in Methods, and on day 7 were tested for morphine-induced antinociception. Mice were placed on a hotplate (52°C) and the time taken for the animals to lick their paws or jump was measured. The animals were tested three times to establish a stable baseline, then injected with 10 mg kg⁻¹ of morphine hydrochloride, and their response latency tested 20 min later. The response latencies (s) (±s.d.) are plotted for each condition; n=10 for vehicle/morphine, n=12 for chronic morphine/morphine. Statistical significance was tested by factorial ANOVA followed by a Scheffe F-test.

Figure 2

DAMGO continues to inhibit I_Ca in PAG neurons from CMT mice. I_Ca was evoked by repetitively stepping PAG neurons from −90 to 0 mV. (a) (i) Time plot of the I_Ca amplitude of a PAG neuron taken from a morphine-treated mouse, illustrating the effect of a range of DAMGO concentrations. Drugs were applied for the duration indicated by the bars. (ii) Example traces from the experiment illustrated in (i). (b) Concentration–response relationship for DAMGO inhibition of I_Ca in PAG neurons from vehicle- and morphine-treated mice. Each point represents the mean±s.e.m. of at least nine cells; curves were fit to the pooled data. The maximum effect of DAMGO was reduced in neurons from CMT animals (P<0.05, two-way ANOVA followed by Bonferroni post hoc test).

Figure 3

Baclofen inhibition of I_Ca in PAG neurons is unaffected by CMT. I_Ca was evoked by repetitively stepping PAG neurons from −90 to 0 mV. Concentration–response relationship for baclofen inhibition of I_Ca in PAG neurons from vehicle- and morphine-treated mice is shown. Each neuron was tested for DAMGO sensitivity before baclofen application and only opioid-sensitive cells were included in the analysis. Each point represents the mean± s.e.m. of at least six cells; curves were fit to the pooled data.

Figure 4

μ-Opioid receptor inhibition of I_Ca is still mediated by G-protein βγ subunits in morphine-treated mice. Example traces of a PAG neuron from a morphine-treated mouse subjected to a conditioning pulse paradigm, which is illustrated beneath the traces. The depolarizing step to +80 mV partially relieved the DAMGO-mediated inhibition of the test step. Note that 45 ms of the conditioning step has been removed from the traces for clarity. These experiments were performed without online leak subtraction, and zero holding current is represented by the dotted line.

Figure 5

Homologous tolerance of opioid-activated potassium conductance after CMT. (a) Example traces of currents from neurons voltage clamped at −56 mV in spontaneously withdrawn slices. Met-enkephalin (30 μM, superfusion shown by bars) induced an outward current in (i) a vehicle-treated neuron and a smaller outward current in (ii) a neuron from a morphine-treated mouse in a slice pretreated with wortmannin (5 μM)/sucrose (50 mM). (b) Average outward current induced by met-enkephalin (30 μM) or baclofen (10 μM) for neurons voltage clamped to −56 mV. The met-enkephalin-induced current was smaller in neurons from dependent mice than in neurons from vehicle mice when GAT-1 activation was prevented or not (no in vitro treatment). GAT-1 activation was blocked by GAT-1 inhibitors (NO-711 10 μM or SKF88976A 10 μM), after wortmannin (5 μM)/sucrose (50 mM) pretreatment and in the presence of Rp-8-CPT-cAMPS (100 μM). All experiments using GAT-1 inhibitors, wortmannin and Rp-8-CPT-cAMPS were performed in the presence of tetrodotoxin (1 μM), CNQX (10 μM), picrotoxin (100 μM) and CGP55845 (1 μM). The number of neurons is shown above the bars and each current amplitude is presented as mean±s.e.m. Statistical significance was tested using a Student's t-test.