Ultrastructural evidence for mu and delta opioid receptors at noradrenergic dendrites and glial profiles in the cat locus coeruleus.

The Locus Coeruleus (LC) is a pontine nucleus involved in many physiological processes, including the control of the sleep/wake cycle (SWC). At cellular level, the LC displays a high density of opioid receptors whose activation decreases the activity of LC noradrenergic neurons. Also, microinjections of morphine administered locally in the LC of the cat produce sleep associated with synchronized brain activity in the electroencephalogram (EEG). Even though much of the research on sleep has been done in the cat, the subcellular location of opioid receptors in the LC and their relationship with LC noradrenergic neurons is not known yet in this species. Therefore, we conducted a study to describe the ultrastructural localization of mu-opioid receptors (MOR), delta-opioid receptors (DOR) and tyrosine hydroxylase (TH) in the cat LC using high resolution electron microscopy double-immunocytochemical detection. MOR and DOR were localized mainly in dendrites (45% and 46% of the total number of profiles respectively), many of which were noradrenergic (35% and 53% for MOR and DOR, respectively). TH immunoreactivity was more frequent in dendrites (65% of the total number of profiles), which mostly also expressed opioid receptors (58% and 73% for MOR and DOR, respectively). Because the distribution of MORs and DORs are similar, it is possible that a substantial sub-population of neurons co-express both receptors, which may facilitate the formation of MOR-DOR heterodimers. Moreover, we found differences in the cat subcellular DOR distribution compared with the rat. This opens the possibility to the existence of diverse mechanisms for opioid modulation of LC activity.

Cellular and subcellular distributions of delta opioid receptor activation sites in the ventral oral pontine tegmentum of the cat.

The ventral division of the reticular oral pontine nucleus (vRPO) is a pontine tegmentum region critically involved in REM sleep generation. Previous reports of morphine microinjections in the cat pontine tegmentum have shown that opioid receptor activation in this region modulates REM sleep. Even though opiate administration has marked effects on sleep-wake cycle architecture, the distribution of opioid receptors in vRPO has only been partially described. Using an antiserum directed against delta opioid receptor (DOR), to which morphine binds, in the present study, we use (1) light microscopy to determine DOR cellular distribution in the rostral pontine tegmentum and (2) electron microscopy to determine DOR subcellular distribution in the cat vRPO. In the dorsal pons, DOR immunoreactivity was evenly distributed throughout the neuropil of the reticular formation and was particularly intense in the parabrachial nuclei and locus coeruleus; the ventral and central areas of the RPO and locus coeruleus complex were especially rich in DOR-labeled somata. Within the vRPO, DOR was localized mainly in the cytoplasm and on plasma membranes of medium to large dendrites (47.8% of DOR-labeled profiles), which received both symmetric and asymmetric synaptic contacts mainly from non-labeled (82% of total inputs) axon terminals. Less frequently, DOR was distributed presynaptically in axon terminals (19% of DOR-labeled profiles). Our results suggest that DOR activation in vRPO regulates REM sleep occurrence by modulating postsynaptic responses to both excitatory and inhibitory afferents. DOR activation in vRPO could have, however, an additional role in direct modulation of neurotransmitter release from axon terminals.

Morphine acutely regulates opioid receptor trafficking selectively in dendrites of nucleus accumbens neurons.

Morphine stimulates the internalization of mu-opioid receptors (MORs) in transfected cell models to a lesser degree than opioid peptides and other analgesic drugs, such as methadone, and previous studies have reported that morphine does not produce a detectable redistribution of MORs in neural tissue after either acute or chronic administration. Nevertheless, morphine produces profound physiological effects, raising the question of whether receptor trafficking plays any role in the in vivo actions of morphine. We investigated the effects of opiate drugs on recombinant and native opioid receptors in the nucleus accumbens, which plays an important role in mediating the behavioral effects of opiate drugs. Morphine and methadone differed in their effects on the internalization of epitope-tagged MORs in cell bodies, introduced by viral gene transfer and imaged by fluorescence microscopy. A mutation of the cytoplasmic tail that confers morphine-induced internalization in cultured cells had a similar effect on receptor trafficking in nucleus accumbens cell bodies. Surprisingly, in contrast to its failure to affect MOR distribution detectably in cell bodies, acute morphine administration produced a pronounced change in MOR distribution visualized in the processes of the same neurons. A similar effect of acute morphine administration was observed for endogenously expressed MORs by immunoelectron microscopy; the acute administration of morphine increased the density of MORs associated with internal membrane structures specifically in dendrites. These results provide the first evidence that morphine regulates the distribution of MORs in neuronal processes, suggesting that "compartment-selective" membrane trafficking represents a previously unanticipated type of opioid receptor regulation contributing to the in vivo effects of opiate drugs on a physiologically relevant population of CNS neurons.

Ultrastructural localization of delta-opioid receptors in the rat caudate-putamen nucleus during postnatal development: relation to synaptogenesis.

During development, delta-opioid receptors (DORs) in the rat caudate-putamen nucleus (CPN) appear later than mu-opioid receptors (MORs), whose developmental pattern specifically relates to synaptogenesis. We used electron microscopic immunocytochemistry to determine whether there are also age-related changes in subcellular localization of DORs in the rat CPN. Sections from postnatal day (P) 0-P30 and adult dorsomedial CPN were immunogold-silver labeled to examine the plasmalemmal and cytoplasmic distribution of these receptors. In addition, immunoperoxidase labeling was used to determine the numerical density of synapses relative to DOR-labeled profiles. Immunolabeling for DOR was undetectable at P0, light at P5, and dense from P10 onward. The labeling during P5-P10 was mainly localized in somatodendritic profiles but also was readily seen in axon terminals, most of which formed asymmetric synapses with dendrites. From P15, a few immunogold particles were seen in contact with postsynaptic densities in spines, and the proportion of these particles significantly increased in P30 and adult CPN. Other particles were localized in the cytoplasm of dendrites and terminals without significant age-related changes. Stereological analysis showed that compared with labeled dendritic shafts and spines, labeled axon terminals have a closer correlation with synapse formation. These results are in marked contrast with MORs, which show an age-related increase in association with dendritic plasma membrane and a good correlation in the developmental pattern of MOR-labeled spines with synapse formation (Wang et al. [2003] Neuroscience 118:695-708). Together, our results suggest receptor-type specific roles for endogenous opioids acting at both pre- and postsynaptic sides in the developing CPN.

Localization and regulation of the delta-opioid receptor in dorsal root ganglia and spinal cord of the rat and monkey: evidence for association with the membrane of large dense-core vesicles.

Using immunohistochemistry and immunoelectron microscopy, the localization and regulation of delta-opioid receptor-like immunoreactivity were studied in dorsal root ganglia and spinal cord of normal rat and monkey, and after peripheral axotomy. Delta-opioid receptor-like immunoreactivity was observed in many small dorsal root ganglion neurons, and in the rat most of them contained substance P and calcitonin gene-related peptide. At the ultrastructural level, delta-opioid receptor-like immunoreactivity was localized in the Golgi complex, on the membrane of the large dense-core vesicles and on the membrane of and/or inside a type of large vesicle with an interior of low electron density. The latter vesicles were often in contact with multivesicular bodies. In the superficial dorsal horn of the spinal cord, most delta-opioid receptor-positive nerve fibers contain substance P and/or calcitonin gene-related peptide, both in rat and monkey. Also, in these nerve endings delta-opioid receptor-like immunoreactivity was found on the membrane of large dense-core vesicles and on the membrane of, or in, the lucent vesicles. Occasionally, delta-opioid receptor-like immunoreactivity was observed on the plasmalemma of the terminals, particularly when the vesicles were in exocytotic contact with the plasmalemma. Peripheral axotomy induced a decrease in delta-opioid receptor-like immunoreactivity both in cell bodies in the dorsal root ganglia and in terminals in the dorsal horn. These data suggest that the delta-opioid receptor may be a constituent of the membrane of large dense-core vesicles storing and releasing neuropeptides. It is suggested that upon exocytotic release of substance P and calcitonin gene-related peptide from large dense-core vesicles, there is a transient modification of the surface of the primary afferent terminals which leads to exposure of the receptor protein so that enkephalin released from adjacent terminals can activate the receptor. The decrease in delta-opioid receptors after axotomy indicates that delta-opioid receptor-mediated inhibitory effects are attenuated at the spinal level both in the rat and monkey.

C1 adrenergic neurons are contacted by presynaptic profiles containing DELTA-opioid receptor immunoreactivity.

Ligands of the delta-opioid receptor tonically influence sympathetic outflow. Some of the actions of delta-opioid receptor agonists may be mediated through C1 adrenergic neurons in the rostral ventrolateral medulla. The goal of this study was to determine whether C1 adrenergic neurons or their afferents contain delta-opioid receptors. Single sections through the rostral ventrolateral medulla were labeled for delta-opioid receptor using the immunoperoxidase method and the epinephrine synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) using the immunogold method, and examined at the light and electron microscopic level. Few ( approximately 5% of 903) profiles dually labeled for PNMT and delta-opioid receptor were detected; most of these were dendrites with diameters < 1.5 microm. delta-Opioid receptor immunoreactivity was affiliated with multivesicular bodies in dually labeled perikarya, whereas delta-opioid receptor immunoperoxidase labeling appeared as isolated clusters within both singly and dually labeled dendrites. The majority ( approximately 83% of 338) of delta-opioid receptor-immunoreactive profiles were axons and axon terminals. delta-Opioid receptor-immunoreactive terminals averaged 0.75 microm in diameter, contained numerous large dense-core vesicles and usually formed appositions or asymmetric (excitatory-type) synapses with their targets. The majority (>50% of 250) of delta-opioid receptor-immunoreactive axons and axon terminals contacted PNMT-immunoreactive profiles. Most of the contacts formed by delta-opioid receptor-immunoreactive profiles ( approximately 75% of 132) were on single-labeled PNMT-immunoreactive dendrites with diameters <1.5 microm. The prominent localization of delta-opioid receptors to dense-core vesicle-rich presynaptic profiles suggests that delta-opioid receptor activation by endogenous or exogenous agonists may modulate neuropeptide release. Furthermore, the presence of delta-opioid receptors on axon terminals that form excitatory-type synapses with PNMT-immunoreactive dendrites suggests that delta-opioid receptor ligands may modulate afferent activity to C1 adrenergic neurons. The observation that some PNMT-immunoreactive neurons contain delta-opioid receptor immunoreactivity associated with multivesicular bodies and other intracellular organelles suggests that some C1 adrenergic neurons may present, endocytose and/or recycle delta-opioid receptors.

Ultrastructural study of delta-opioid receptors in the dorsal horn of the rat spinal cord using monoclonal anti-idiotypic antibodies.

The ultrastructural localization of delta-opioid receptors was studied using monoclonal anti-idiotypic antibody prepared with an anti-D-Ala2-D-Leu5-enkephalin. Immunocytochemical techniques were used on vibratome sections from rats perfused with paraformaldehyde. A high density of immunoreactivity was observed in the dorsal horn of the spinal cord, particularly the two superficial layers, the dorsolateral funiculus and the area surrounding the central canal. The labelling was absent when the antibody was preincubated with the immunogen. Competition between the anti-idiotypic antibody and different ligands, delta or mu, was controlled by preincubation of tissue sections with the ligand in the presence of peptidase inhibitors for 3-4 h before addition of the anti-idiotypic antibody. Enkephalin, dermenkephalin and naltrindole induced disappearance of the labelling at 10(-9) M while dermorphin or dermorphin Lys7 were ineffective at the same concentration. Lamina II of the dorsal horn was studied by electron microscopy. The immunolabelling was mainly localized on cell membranes at appositions between the two neurons. About one third were localized between an axon terminal and a dendrite, the same proportion of labellings were between two axon terminals. Labelling was occasionally observed at appositions between a glomerular terminal and a dendrite or a terminal or at axoglial appositions. Axosomatic localizations were rare. The presynaptic localization of the labelling is in favor of a presynaptic mechanism of action for delta-opioids in the spinal cord, providing that these receptors are functional. delta-Opioid peptides probably act non-synaptically since receptors were never localized on synaptic differentiations.

Dual ultrastructural immunocytochemical labeling of mu and delta opioid receptors in the superficial layers of the rat cervical spinal cord.

The delta opioid receptor (DOR) and mu opioid receptor (MOR) are abundantly distributed in the dorsal horn of the spinal cord. Simultaneous activation of each receptor by selective opiate agonists has been shown to result in synergistic analgesic effects. To determine the cellular basis for these functional associations, we examined the electron microscopic immunocytochemical localization of DOR and MOR in single sections through the superficial layers of the dorsal horn in the adult rat spinal cord (C2-C4). From a total of 270 DOR-labeled profiles, 49% were soma and dendrites, 46% were axon terminals and small unmyelinated axons, and 5% were glial processes. 6% of the DOR-labeled soma and dendrites, and < 1% of the glial processes also showed MOR-like immunoreactivity (MOR-LI). Of 339 MOR-labeled profiles, 87% were axon terminals and small unmyelinated axons, 12% were soma and dendrites, and 2% were glial processes. 21% of the MOR-labeled soma and dendrites, but none of the axon terminals also contain DOR-LI. The subcellular distributions of MOR and DOR were distinct in axon terminals. In axon terminals, both DOR-LI and MOR-LI were detected along the plasmalemma, but only DOR-LI was associated with large dense core vesicles. DOR-labeled terminals formed synapses with dendrites containing MOR and conversely, MOR-labeled terminals formed synapses with DOR-labeled dendrites. These results suggest that the synergistic actions of selective MOR- and DOR-agonists may be attributed to dual modulation of the same or synaptically linked neurons in the superficial layers of the spinal cord.

The influences of reproductive status and acute stress on the levels of phosphorylated delta opioid receptor immunoreactivity in rat hippocampus.

In the hippocampus, ovarian hormones and sex can alter the trafficking of delta opioid receptors (DORs) and the proportion of DORs that colocalize with the stress hormone, corticotropin releasing factor. Here, we assessed the effects of acute immobilization stress (AIS) and sex on the phosphorylation of DORs in the rat hippocampus. We first localized an antibody to phosphorylated DOR (pDOR) at the SER363 carboxy-terminal residue, and demonstrated its response to an opioid agonist. By light microscopy, pDOR-immunoreactivity (ir) was located predominantly in CA2/CA3a pyramidal cell apical dendrites and in interneurons in CA1-3 stratum oriens and the dentate hilus. By electron microscopy, pDOR-ir primarily was located in somata and dendrites, associated with endomembranes, or in dendritic spines. pDOR-ir was less frequently found in mossy fibers terminals. Quantitative light microscopy revealed a significant increase in pDOR-ir in the CA2/CA3a region of male rats 1h following an injection of the opioid agonist morphine (20mg/kg, I.P). To look at the effects of stress on pDOR, we compared pDOR-ir in males and cycling females after AIS. The level of pDOR-ir in stratum radiatum of CA2/CA3a was increased in control estrus (elevated estrogen and progesterone) females compared to proestrus and diestrus females and males. However, immediately following 30min of AIS, no significant differences in pDOR levels were seen across estrous cycle phase or sex. These findings suggest that hippocampal levels of phosphorylated DORs vary with estrous cycle phase and that acute stress may dampen the differential effects of hormones on DOR activation in females.