Presynaptic, but not postsynaptic, MORs resist acute desensitization
To examine the desensitization of pre- and postsynaptic MORs during exposure to ME, whole cell voltage clamp recordings were made from fluorescently labeled POMC neurons. Presynaptic and postsynaptic effects of ME were measured simultaneously. The MOR selective opioid agonist ME (30 μM) induced a postsynaptic outward current (42 ± 14 pA, n = 6; ) that desensitized significantly within minutes of continued exposure (p < 0.001, one-way repeated measures ANOVA, τ = 116 s, plateau = 46% of baseline; ). ME also caused a robust inhibition of the amplitude of eIPSCs (to 24 ± 3.5% of baseline, n = 14, p < 0.0001; ). Inhibition of eIPSC amplitude was maintained throughout the superfusion of ME (30 μM; p = 0.27, one-way repeated measures ANOVA, minutes 2 through 10; ). Together these results indicate that postsynaptic, but not presynaptic, MORs regulating POMC neurons acutely desensitize when exposed to a maximal concentration of ME similar to previous results with the agonist [D-Ala2
]-enkephalin (DAMGO, (Pennock and Hentges, 2011
Postsynaptic, but not presynaptic, MORs desensitize acutely
Presynaptic resistance to desensitization is a property of multiple Gi/o-coupled receptors
To determine if differential pre- and postsynaptic desensitization may be a general property of Gi/o-coupled receptors, or rather a specific property of MORs, the ability of GABAB, nociceptin, and kappa opioid receptors (KORs) to undergo or resist desensitization was examined. Superfusion of a maximal concentration of nociceptin (500 nM) induced a postsynaptic outward current (38 ± 4.8 pA peak) that declined within minutes to a plateau of 40% of baseline (p < 0.0001, one-way repeated measures ANOVA, τ = 210 s, n=6; ). A maximal concentration of nociceptin (500 nM) also caused an inhibition of eIPSC amplitude (to 30 ± 7.7% of baseline, n = 7; ) but the inhibition of eIPSCs did not decline during the 10 min exposure to agonist (p = 0.47, one-way repeated measures ANOVA, minutes 2 through 10; ). Similarly, the GABABR agonist baclofen (30 μM, maximal concentration) induced an outward current (36 ± 6.0 pA, n = 6; ) that declined during exposure (plateau = 53% of baseline, p < 0.0001, one-way repeated measure ANOA, τ = 84 s; ). Baclofen also caused an inhibition of eIPSC amplitude (to 36 ± 3.4% of baseline, n = 19; ) that was maintained throughout the exposure (p = 0.053, one-way repeated measures ANOVA, minutes 2 through 10; ).
Multiple Gi/o-coupled receptors presynaptic to POMC neurons resist acute desensitization
Activation of the KOR inhibits neurotransmitter release onto POMC neurons, but has no apparent postsynaptic effects (Pennock and Hentges, 2011
). Superfusion of a maximal concentration of the KOR agonist U69593 (500 nM) resulted in inhibition of the amplitude of eIPSCs (to 66 ± 9.3% of baseline, n = 5, p < 0.0001; ). Similar to the MOR, GABAB
R, and nociceptin receptors, the KOR-mediated inhibition of eIPSC amplitude showed no desensitization during >10 min exposure to U69593 (p = 0.071, one-way repeated measures ANOVA, minutes 3 through 13, n = 5; ). Nor-BNI (100 nM) was added upon the termination of U69593 superfusion to enhance wash/reversal (). Together these data show that resistance to acute desensitization may be a common characteristic of Gi/o
-coupled receptors located on presynaptic terminals.
Presynaptic MORs resist desensitization when activated with morphine
Differential desensitization of pre- and postsynaptic Gi/o-coupled receptors in POMC neurons
It is plausible that the cell type on which a receptor is located, not the sub-cellular compartment in which it is found, determines whether or not that receptor will be resistant to desensitization. To determine if receptors located in pre- and postsynaptic compartments of a single cell type show differential acute desensitization, experiments were performed where desensitization of presynaptic receptors within POMC neurons was studied using an optogenetic approach. A viral vector containing a floxed ChR2 sequence was injected into the arcuate nucleus of mice expressing Cre recombinase under the control of the POMC promoter. In slices prepared from these mice, brief pulses of blue light depolarized the POMC neurons resulting in the release of neurotransmitter onto unidentified neighboring neurons from which recordings were made. Superfusion of ME (30 μM) resulted in a robust inhibition of light-evoked IPSC amplitude (to 36 ± 10% of baseline, n = 6, p < 0.0001; ), which was reversed by the MOR antagonist CTAP (500 nM). Inhibition of the light-evoked IPSCs was maintained during the exposure to ME (p = 0.14, one-way repeated measures ANOVA, minutes 3 through 10, n = 6; ) indicating that unlike postsynaptic MORs in POMC neurons, MORs directly regulating transmitter release from POMC neurons do not undergo acute desensitization.
Gi/o-coupled receptors on the axon terminals of POMC neurons resist desensitization
Baclofen (30 μM) also inhibited presynaptic release from POMC terminals as indicated by the reduction in light-evoked IPSC amplitude (to 27 ± 7.3% of baseline, n = 6, p < 0.0001; ). This inhibition was maintained during continued agonist exposure (p = 0.051, one-way repeated measures ANOVA, minutes 2 through 10, n = 6; ). Thus, both mu opioid and GABAB receptors undergo desensitization in the postsynaptic ( & ), but not presynaptic compartments ().
Although KOR activation does not induce a GIRK-mediated current in POMC neurons (Pennock and Hentges, 2011
), the KOR agonist U69593 (500 nM) significantly inhibited the amplitude of light-evoked IPSCs (to 45 ± 14% of baseline, n = 4, p < 0.0001; ). The inhibition of transmitter release was maintained during 14 minutes of continuous U69593 perfusion (p = 0.94, one-way repeated measures ANOVA, minutes 4 through 14, n = 4; ). The inhibition of light-evoked IPSC amplitude was completely reversed by the KOR selective antagonist nor-NBI (100 nM; ). Together the data from the light-evoked release studies demonstrate that the ability of some receptors to resist desensitization is likely dependent on the location of the receptor within the neuron.
Decreasing receptor number does not induce presynaptic desensitization
To determine if receptor reserve may account for the lack of desensitization observed for MORs, the irreversible MOR antagonist β-CNA was used to reduce receptor reserve. β-CNA (50 nM) was superfused over the slice for 2 min following the washout of a brief application of ME (30 μM; ). The pulse of ME before β-CNA application was used to determine the maximal inhibition possible for ME before receptor reserve was reduced. After the superfusion of β-CNA and a washout period (>10 min), ME (30 μM) was again applied. β-CNA reduced the inhibition of eIPSC amplitude caused by ME to 60 ± 11% of its original value (83 ± 3.4% inhibition of eIPSC amplitude before β-CNA vs. 49 ± 8.0% inhibition of eIPSC amplitude after β-CNA, n = 3, p = 0.039, paired t-test; ). Although β-CNA treatment reduced the inhibition of eIPSC amplitude by ME, there was still no decrease in ME mediated inhibition (desensitization) over the course of the drug application (p = 0.065, n = 3, one-way repeated measures ANOVA, minutes 2 through 11; ).
Decreasing the number of functional receptors presynaptic to POMC neurons does not induce acute desensitization
To estimate the size of the MOR receptor reserve presynaptic to POMC neurons, dose response curves were constructed before and after β-CNA treatment and analyzed using Furchgott’s method (Furchgott, 1966
); ). ME (30 μM) caused a much larger inhibition of eIPSC amplitude in control conditions compared to the inhibition after a 2 min exposure to β-CNA (79 ± 2.1% inhibition of eIPSC amplitude before β-CNA vs. 42 ± 2.4% inhibition of eIPSC amplitude after β-CNA, p < 0.0001; n=36,12; ). Additionally, there was ~3-fold shift in the EC50
for ME after β-CNA (444 nM before β-CNA vs. 1.336 μM after β-CNA). To estimate the fraction of the total number of presynaptic receptors inhibited by β-CNA a double reciprocal plot of the agonist concentration needed to achieve a given inhibition of eIPSC amplitude before and after β-CNA treatment was constructed according to Furchgott’s method (Equation 1
). This analysis revealed that the 44% decrease in the maximal inhibition of eIPSC amplitude by ME corresponded to a 59–68% reduction in the total number of presynaptic MORs (95% confidence interval for q [0.3189,0.4137]; ) and provided an estimated KA
value near that of the EC50
(95% confidence interval [0.693 μM, 1.315 μM]; ) under control conditions. These data suggest that presynaptic resistance to desensitization is not due to receptor reserve since removing enough receptors to potently blunt the maximal response did not cause presynaptic receptors to display desensitization. Further, the finding that the removing ~50% of the surface receptors, reduces the function response by ~50% indicates that terminals regulating POMC neurons do not have a significant receptor reserve even under baseline conditions.
Morphine does not cause desensitization of MORs in the presynaptic compartment
MORs activated by morphine may desensitize through a mechanism distinct from those activated by ME or DAMGO in some systems (Johnson et al., 2006
; Kelly et al., 2008
). Thus, it is possible that the environment of the presynaptic terminal may confer resistance to desensitization to a ME activated MOR but not a morphine activated receptor. To determine if resistance to desensitization by presynaptic MORs may be agonist-specific, eIPSC amplitude was measured during a 15 min superfusion of morphine (20 μM). Morphine caused a reduction in the amplitude of eIPSCs (to 52 ± 12% of baseline, n=4, p < 0.0001) that was maintained throughout exposure to morphine (p = 0.91, n = 4, one-way repeated measures ANOVA, minutes 3 through 13; ) indicating that presynaptic receptors do not undergo acute desensitization whether bound by full or partial agonists that likely confer different conformational states of the receptor.
Chronic morphine treatment (CMT) can reduce the efficacy of MOR agonists at presynaptic terminals (North and Vitek, 1980
; Fyfe et al., 2010
) and enhance the extent and rate of acute desensitization of postsynaptic MORs (Dang and Williams, 2005
; Ingram et al., 2008
). To determine if CMT can modify presynaptic MOR signaling to enhance acute desensitization of these receptors in terminals presynaptic to POMC neurons, mice were treated with morphine (50 mg/kg/day) or saline for 5–7 days using subcutaneous mini-osmotic pumps prior to slice preparation. When slices were collected into morphine (1 μM) and maintained in morphine, application of naloxone (1 μM) caused a greater increase in the eIPSC amplitude (1.8 ± 0.13 fold increase in control, 4.4 ± 1.0 fold increase in CMT, n=4–6, p=0.01, ) and decrease in the paired-pulse ratio in cells from CMT mice compared to saline-treated mice (0.68 ± 0.04 in control, 0.51 ± 0.05 in CMT, n=4–6, p=0.01; ), indicating that morphine was effectively reaching the synapses and inhibiting transmitter release. Application of ME (30 μM) to slices from CMT mice that were collected and maintained in morphine-free aCSF resulted in a robust decrease in eIPSC amplitude (to 32 ± 3.2 % of baseline, n = 20, p < 0.0001; ) that was maintained over the course of an 11 min exposure (p = 0.32, n = 5, one-way repeated measures ANOVA, minutes 2 through 11) similar to that observed for tissue from untreated mice. Thus, presynaptic MORs resist desensitization after both acute and chronic exposure to morphine.
Chronic morphine treatment does not enhance desensitization of presynaptic MORs
A fraction of presynaptic GABABRs acutely desensitize
It is plausible that a property of the presynaptic environment could somehow prevent receptor desensitization. However, in 7 of 26 recordings made from POMC neurons where baclofen (30 μM) was applied while recording eIPSC amplitude, the inhibition of eIPSC amplitude declined during baclofen application (p < 0.0001, 1 way repeated measures ANOVA, minutes 2 through 10, n = 7; ). Inhibition peaked by the 2nd minute of agonist exposure on average, and desensitized by ~50% by minute 10 (25 ± 6.9% of baseline at minute 2 vs. 60 ± 7.4% of baseline at minute 10). In some recordings where GABAB receptor desensitization was observed, it was possible to subsequently perfuse a maximal concentration of ME for >10 minutes. In these recordings, only the GABAB-mediated inhibition of eIPSC amplitude desensitized whereas ME-mediated inhibition was maintained (data not shown). Additionally, GABAB mediated inhibition of miniature IPSC (mIPSC) frequency was examined to determine if ~25% of all inputs onto POMC neurons contain GABAB receptors which are able to undergo desensitization, or if only ~25% of POMC neurons receive inputs with GABAB receptors that are able to undergo desensitization. Similar to what was observed when using eIPSCs to measure GABAB mediated inhibition of neurotransmitter release, desensitization of GABAB mediated inhibition of mIPSC frequency was only observed in 25% of recordings made (3 of 12 recordings, ). This suggests that 25% of POMC neurons receive input from terminals on which GABAB receptors are able to undergo desensitization. If resistance or susceptibility of GPCRs to desensitization is due to a property of the terminals on which they are found desensitization should have been observed in a similar fraction of recordings using agonists for the other receptors examined. Altogether, it appears that resistance to desensitization is likely conferred by a property of the receptor itself and not an intrinsic property of the terminals on which the receptors are found.
A fraction of GABABRs presynaptic to POMC neurons do desensitize