Desensitization of Mu Opioid Receptors in the PreBö tzinger Complex.

Lowe JD, Kelly E, Henderson G. University of Bristol, Bristol, UK

A major adverse consequence of the use of opiates is respiratory depression which if severe enough can lead to death. Opiate users have an increased risk of overdose death after periods of abstinence, such as release from prison or treatment programs, suggesting that recovery from tolerance to the respiratory depressant effects of opiates has made them more vulnerable. Opioid agonists suppress respiration by activating opioid receptors in the ventral respiratory column and more specifically within glutamatergic neurones of the preBötzinger complex (preBötC) which is believed to be the dominant respiration rhythm generator. The preBötC contains a heterogeneous population of neurones, but cells coexpressing mu-opioid receptors (MOPr) and neurokinin-1 receptors (NK1) are thought to generate the inspiratory rhythm. In this study, we sought to determine if MOPrs within the preBötC undergo agonist-dependent desensitization using whole-cell patch-clamp electrophysiology in mouse medullary brain slices.

Coronal brain slices (250 micron thick) containing the preBötC were prepared from 4-5 week old male CD1 mice. Postsynaptic opioid receptor responses were measured as either an outward current in neurones voltage clamped at -60 mV or as a hyperpolarization in neurones current clamped to a membrane potential of -45 mV. Presynaptic opioid receptor responses were measured in neurones voltage clamped at -60 mV as a decrease in the amplitude of pharmacologically isolated glutamatergic evoked excitatory postsynaptic currents (eEPSCs) evoked by a locally placed bipolar stimulating electrode. Neurones were also assessed for postsynaptic NK1 receptor and GABA-B receptor responses.

67% of neurones (n=36) showed a postsynaptic response to a receptor saturating concentration of Met-enkephalin (30 microM) which was measured either as an outward current of 33.5 ± 4.4 pA or as a hyperpolarization of 8.2 ± 2.8 mV. Of the Met-enkephalin responding cells, 57% showed an inward current in response to the NK1 agonist [Sar9, Met(O2)11]-Substance P (500 nM) and 81% produced an outward current in response to the GABA-B agonist baclofen (20 microM). The postsynaptic Met-enkephalin responses desensitized by 57 ± 6% (n=6) after 10 min of agonist application. We next examined whether presynaptic MOPrs within the preBötC inhibited glutamatergic transmission. Met-Enkephalin (30 microM) produced a 60 ± 11% inhibition of the eEPSC amplitude (n=5), however this inhibition remained stable over a 10 min application (63 ± 8% inhibition at end of application) and was reversed by either the non-selective opioid antagonist naloxone (1 microM) or the MOPr selective antagonist CTAP (1 microM) . Morphine (30 microM) produced a 45 ± 8% inhibition of eEPSC amplitude (n=6), that also did not desensitize over a 10 min application (56 ± 9% inhibition at end of application) and was reversed by CTAP. The delta opioid receptor selective agonist DPDPE (10 microM) did not produce a significant inhibition of the eEPSC amplitude (8.5 ± 4.4%, n=4).

Thus, in the preBötC postsynaptic MOPrs exhibit agonist-dependent desensitization whereas presynaptic MOPrs do not undergo rapid agonist-induced desensitization. Further studies will examine whether tolerance develops to the MOPr effects in the preBötC after chronic in vivo treatment with opiates.