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Dorsal horn neurons send ascending projections to both thalamic nuclei and parabrachial nuclei; these pathways are thought to be critical pathways for central processing of nociceptive information. Afferents from the corneal surface of the eye mediate nociception from this tissue which is susceptible to clinically important pain syndromes. This study examined corneal afferents to the trigeminal dorsal horn and compared inputs to thalamic- and parabrachial-projecting neurons. We used anterograde tracing with cholera toxin B subunit to identify corneal afferent projections to trigeminal dorsal horn, and the retrograde tracer FluoroGold to identify projection neurons. Studies were conducted in adult male Sprague-Dawley rats. Our analysis was conducted at two distinct levels of the trigeminal nucleus caudalis (Vc) which receive corneal afferent projections. We found that corneal afferents project more densely to the rostral pole of Vc than the caudal pole. We also quantified the number of thalamic- and parabrachial-projecting neurons in the regions of Vc that receive corneal afferents. Corneal afferent inputs to both groups of projection neurons were also more abundant in the rostral pole of Vc. Finally, by comparing the frequency of corneal afferent appositions to thalamic- versus parabrachial-projecting neurons, we found that corneal afferents preferentially target parabrachial-projecting neurons in trigeminal dorsal horn. These results suggest that nociceptive pain from the cornea may be primarily mediated by a non-thalamic ascending pathway., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

The periaqueductal gray (PAG) is part of a descending pain modulatory system that, when activated, produces widespread and profound antinociception. Microinjection of either opioids or cannabinoids into the PAG elicits antinociception. Moreover, microinjection of the cannabinoid 1 (CB1) receptor agonist HU-210 into the PAG enhances the antinociceptive effect of subsequent morphine injections, indicating a direct relationship between these two systems. The objective of this study was to characterize the distribution of CB1 receptors in the dorsolateral and ventrolateral PAG in relationship to mu-opioid peptide (MOP) receptors. Immunocytochemical analysis revealed extensive and diffuse CB1 receptor labeling in the PAG, 60% of which was found in somatodendritic profiles. CB1 and MOP receptor immunolabeling were co-localized in 32% of fluorescent Nissl-stained cells that were analyzed. Eight percent (8%) of PAG neurons that were MOP receptor-immunoreactive (-ir) received CB1 receptor-ir appositions. Ultrastructural analysis confirmed the presence of CB1 receptor-ir somata, dendrites and axon terminals in the PAG. These results indicate that behavioral interactions between cannabinoids and opioids may be the result of cellular adaptations within PAG neurons co-expressing CB1 and MOP receptors., (Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.)

Microinjection of opioids into the ventrolateral periaqueductal gray (vlPAG) produces antinociception in part by binding to mu-opioid receptors (MOPrs). Although both high and low efficacy agonists produce antinociception, low efficacy agonists such as morphine produce limited MOPr internalization suggesting that MOPr internalization and signaling leading to antinociception are independent. This hypothesis was tested in awake, behaving rats using DERM-A594, a fluorescently labeled dermorphin analog, and internalization blockers. Microinjection of DERM-A594 into the vlPAG produced both antinociception and internalization of DERM-A594. Administration of the irreversible opioid receptor antagonist beta-chlornaltrexamine (beta-CNA) prior to DERM-A594 microinjection reduced both the antinociceptive effect and the number of DERM-A594 labeled cells demonstrating that both effects are opioid receptor-mediated. Pretreatment with the internalization blockers dynamin dominant-negative inhibitory peptide (dynamin-DN) and concanavalinA (ConA) attenuated both DERM-A594 internalization and antinociception. Microinjection of dynamin-DN and ConA also decreased the antinociceptive potency of the unlabeled opioid agonist dermorphin when microinjected into the vlPAG as demonstrated by rightward shifts in the dose-response curves. In contrast, administration of dynamin-DN had no effect on the antinociceptive effect of microinjecting the GABA(A) receptor antagonist bicuculline into the vlPAG. The finding that dermorphin-induced antinociception is attenuated by blocking receptor internalization indicates that key parts of opioid receptor-mediated signaling depend on internalization., (2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Endomorphins represent a group of endogenous opioid peptides with high affinity for the mu-opioid receptor. In the brainstem, Endomorphin-2 is found in trigeminal dorsal horn and the nuclei of the solitary tract, suggesting its presence in both nociceptive and visceral primary afferents. If Endomorphin-2 were an endogenous ligand for the mu-opioid receptor, we would expect to find the receptor at cellular sites in close association with the peptide. We used dual-labeling immunocytochemistry combined with electron microscopy to examine interactions between Endomorphin-2-immunoreactive and mu-opioid receptor-immunoreactive profiles within the nuclei of the solitary tract in the rat. Endomorphin-2-immunoreactivity was found primarily in unmyelinated axons and axon terminals in nuclei of the solitary tract and the majority of these terminals contained dense core vesicles. Endomorphin-2-immunoreactive axon terminals often formed asymmetric synapses with dendritic spines lacking mu-opioid receptor-immunoreactivity, but mu-opioid receptor-immunoreactivity was found in many of the larger dendritic targets of Endomorphin-2-immunoreactive terminals. Thus, mu-opioid receptor-immunoreactivity was found in the postsynaptic targets of Endomorphin-2-immunoreactive axon terminals, consistent with the hypothesis that Endomorphin-2 is an endogenous ligand for this receptor within the nuclei of the solitary tract. A small number of Endomorphin-2-immunoreactive somata, dendrites, and axon terminals also contained mu-opioid receptor-immunoreactivity. Cells that contain both the opioid peptide and its receptor may be a substrate for potential autoregulation of nuclei of the solitary tract neurons by opioid ligands. Finally, using tract tracing and confocal microscopy, we found Endomorphin-2-immunoreactivity in a subset of vagal afferents. Together these findings support the hypothesis that Endomorphin-2 is a ligand for the mu-opioid receptor within nuclei of the solitary tract and that the peptide is at least partially derived from primary visceral afferents.

Ligands of the mu-opioid receptor are known to inhibit nociceptive transmission in the dorsal horn, yet the cellular site(s) of action for this inhibition remain to be fully elucidated. Neurons located in lamina I of the dorsal horn are involved in distinct aspects of nociceptive transmission. Neurons projecting to the thalamus are thought to be involved in sensory-discriminative aspects of pain perception, while neurons projecting to the parabrachial nucleus are thought to be important for emotional and/or autonomic responses to noxious stimuli. The present study examined these two populations of lamina I projection neurons in the trigeminal dorsal horn to determine if the mu-opioid receptor protein (MOR1) is differentially located in these populations of neurons. Lamina I projection neurons were identified using the retrograde tracer FluoroGold (FGold). FGold was injected into either the contralateral thalamus (ventral posterolateral (VPM)/ventral posterolateral (VPL) thalamic region) or into the ipsilateral parabrachial nuclei. The distribution of MOR1 in these neurons was determined using immunocytochemistry. The distribution of MOR1-ir within these two populations of lamina I projection neurons was examined by both confocal and electron microscopy. We found that both populations of projection neurons contained MOR1. Immunogold analyses revealed the presence of MOR1-ir at membrane sites and within the cytoplasm of these neurons. Cytoplasmic receptor labeling may represent sites of synthesis, recycling or reserve populations of receptors. MOR1 was primarily found in the somata and proximal dendrites of projection neurons. In addition, these neurons rarely received synaptic input from MOR1-containing axon terminals. These results indicate that lamina I neurons in trigeminal dorsal horn that project to the thalamic and parabrachial nuclei contain MOR1 and are likely sites of action for MOR ligands that modulate sensory and/or autonomic aspects of pain transmission in the trigeminal dorsal horn.

Activation of opioid receptors in the CNS evokes a dramatic decrease in heart rate which is mediated by increases in inhibitory parasympathetic activity to the heart. Injection of opiates into the nucleus ambiguus, where premotor cardiac parasympathetic nucleus ambiguus neurons are located elicits an increase in parasympathetic cardiac activity and bradycardia. However, the mechanisms responsible for altering the activity of premotor cardiac parasympathetic nucleus ambiguus neurons is unknown. This study examined at the electron microscopic level whether premotor cardiac parasympathetic nucleus ambiguus neurons possess postsynaptic opioid receptors and whether mu-opioid receptor agonists alter voltage-gated calcium currents in these neurons. Premotor cardiac parasympathetic nucleus ambiguus neurons were identified in the rat using retrograde fluorescent tracers. One series of experiments utilized dual-labeling immunocytochemical methods combined with electron microscopic analysis to determine if premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. In a second series of experiments whole cell patch clamp methodologies were used to determine whether activation of postsynaptic opioid receptors altered voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons in brainstem slices. The perikarya and 78% of the dendrites of premotor cardiac parasympathetic nucleus ambiguus neurons contain mu-opioid receptors. Voltage-gated calcium currents in premotor cardiac parasympathetic nucleus ambiguus neurons were comprised nearly entirely of omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents. Activation of mu-opioid receptors inhibited these voltage-gated calcium currents and this inhibition was blocked by pretreatment with pertusis toxin. The mu-opioid receptor agonist endomorphin-1, but not the mu-opioid receptor agonist endomorphin-2, inhibited the calcium currents. In summary, mu-opioid receptors are located postsynaptically on premotor cardiac parasympathetic nucleus ambiguus neurons. The mu-opioid receptor agonist endomorphin1 inhibited the omega-agatoxin-sensitive P/Q-type voltage-gated calcium currents in premotor cardiac vagal nucleus ambiguus neurons. This inhibition is mediated via a G-protein mediated pathway which was blocked by pretreatment with pertusis toxin. It is possible that the inhibition of calcium currents may act to indirectly facilitate the activity of premotor cardiac parasympathetic nucleus ambiguus neurons by disinhibition, such as by a reduction in inhibitory calcium activated potassium currents., (Copyright 2003 IBRO)

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.

Agonists of the mu-opioid receptor (MOR) can modulate the activity of visceral premotor neurons, including cardiac premotor neurons. Neurons in brainstem regions containing these premotor neurons also contain dense concentrations of the MOR1. This study examined the distribution of MOR1 within two populations of visceral premotor neurons: one located in the dorsal motor nucleus of the vagus and the other in the nucleus ambiguus. Visceral premotor neurons contained the retrograde tracer Fluoro-Gold following injections of the tracer into the pericardiac region of the thoracic cavity. MOR1 was localized using immunogold detection of an anti-peptide antibody. Visceral premotor neurons in both regions contained MOR1 at somatic and dendritic sites, although smaller dendrites were less likely to contain the receptor than larger dendrites, suggesting there may be selective trafficking of MOR1 within these neurons. MOR1 labeling in nucleus ambiguus neurons was more likely to be localized to plasma membrane sites, suggesting that ambiguus neurons may be more responsive to opioid ligands than neurons in the dorsal motor nucleus of the vagus. In addition, many of the dendrites of visceral premotor neurons were in direct apposition to other dendrites. MOR1 was often detected at these dendro-dendritic appositions that may be gap junctions. Together these findings indicate that the activity of individual visceral premotor neurons, as well as the coupling between neurons, may be regulated by ligands of the MOR.

Opioids inhibit nociceptive transmission at the level of the spinal cord, possibly through inhibition of neurotransmitter release by presynaptic mu opioid receptors (MORs) thus preventing the activation of ascending pathways and the perception of pain. Most nociceptive primary afferents are unmyelinated fibers containing peptides such as substance P and/or calcitonin gene-related peptide. However, few terminals contain both substance P and MOR. Recently, we identified new carboxy-terminal MOR splice variants that are localized in the superficial laminae of the dorsal horn. We now report the precise cellular distribution of two of these MOR-1 variants, MOR-1C (exon 7/8/9 epitope) and MOR-1D (exon 8/9 epitope), at the ultrastructural level. In the superficial laminae of the dorsal horn, the majority of the labeling of MOR-1C and MOR-1D was found in unmyelinated axons. This distribution contrasts with that of MOR-1 (exon 4 epitope), in which labeling is equally found in dendrites and soma, as well as in axons. The presence of dense core vesicles in many of the MOR-1C-like immunoreactive terminals implies that this splice variant might be involved in presynaptic inhibition of transmitter release from peptide-containing afferents to the dorsal horn. Consistent with this finding, confocal microscopy analyses showed that many MOR-1C profiles in laminae I-II also contained calcitonin gene-related peptide, whereas fewer MOR-1 profiles contained either substance P or calcitonin gene-related peptide in this same region. From these findings we suggest that there are differential distributions of MOR-1 splice variants as well as distinct peptide colocalizations in the dorsal horn.

The fundamental neuronal substrates of the arterial baroreceptor reflex have been elucidated by combining anatomical, neurophysiological, and pharmacological approaches. A serial pathway between neurons located in the nuclei of the solitary tract (NTS), the caudal ventrolateral medulla (CVL), and the rostral ventrolateral medulla (RVL) plays a critical role in inhibition of sympathetic outflow following stimulation of baroreceptor afferents. In this paper, we summarize our studies using tract-tracing and electron microscopic immunocytochemistry to define the potential functional sites for synaptic transmission within this circuitry. The results are discussed as they relate to the literature showing: (1) baroreceptor afferents excite second-order neurons in NTS through the release of glutamate; (2) these NTS neurons in turn send excitatory projections to neurons in the CVL; (3) GABAergic CVL neurons directly inhibit RVL sympathoexcitatory neurons; and (4) activation of this NTS-->CVL-->RVL pathway leads to disfacilitation of sympathetic preganglionic neurons by promoting withdrawal of their tonic excitatory drive, which largely arises from neurons in the RVL. Baroreceptor control may also be regulated over direct reticulospinal pathways exemplified by a newly recognized sympathoinhibitory region of the medulla, the gigantocellular depressor area. This important autonomic reflex may also be influenced by parallel, multiple, and redundant networks.

N-Methyl-D-aspartate receptors are present in the nodose ganglion, which contains the cell bodies of vagal afferents, and in the nucleus tractus solitarius, where these afferent fibers terminate. This suggests that N-methyl-D-aspartate receptors are located presynaptically on visceral vagal afferents and/or their target neurons in the nucleus tractus solitarius. To test this hypothesis, we combined anterograde transport of biotinylated dextran amine, following injections into the left nodose ganglion, with electron microscopic immunogold labeling of antipeptide antiserum against the R1 subunit of the N-methyl-D-aspartate receptor in the nucleus tractus solitarius of rat brain. Within the medial nucleus tractus solitarius, the N-methyl-D-aspartate receptor R1 immunoreactivity was seen in dendrites (39% of 639 profiles), axons and axon terminals (41%), and a few neuronal perikarya and glia. Many vagal afferent axons and terminals (40% of 468 profiles) contained N-methyl-D-aspartate receptor R1 immunogold labeling. In addition, 42% of the dendrites contacted by vagal afferent terminals (n = 206) contained N-methyl-D-aspartate receptor R1 immunoreactivity. In axons and dendrites, the gold particles were occasionally seen within asymmetric postsynaptic junctions or at non-synaptic sites on the plasma membrane. More commonly, however, N-methyl-D-aspartate receptor R1 labeling was seen on membranes of vesicular cytoplasmic organelles, suggesting that there is abundant N-methyl-D-aspartate receptor protein available for activity-dependent mobilization to the plasmalemma. Since many vagal afferents are glutamatergic, our results implicate N-methyl-D-aspartate receptors in autoregulation of the presynaptic release and postsynaptic responses to glutamate at the level of the first central synapse in the nucleus tractus solitarius.

Several lines of clinical evidence suggest that myoclonus is caused by a reduction of serotonin in the brain and hyperactivity of the inferior olive. We determined whether a change in serotonin content within the olivocerebellar system accompanied a predisposition to myoclonus and investigated the necessity of the inferior olive for a myoclonic seizure. The experiments employed the genetically epilepsy-prone rat that exhibits a profound myoclonic seizure in response to an auditory stimulus. We found that these animals demonstrated a significant reduction in the serotonergic innervation of the inferior olive without a significant change in the serotonergic innervation at any other level of the olivocerebellar circuit. The deficit in olivary serotonin was verified physiologically and pharmacologically by a reduced sensitivity of the genetically epilepsy-prone rat to the tremorogenic effect of harmaline, which is known to produce tremor through a mechanism that requires serotonergic innervation of the inferior olive. We quantified the timing of the myoclonic seizure of the genetically epilepsy-prone rat and found that its large amplitude 2-6 Hz clonus was always preceded by 9-10 Hz tremor that was synchronized among limbs. Ablation of the inferior olive by 3-acetylpyridine abolished the myoclonic seizure. The specificity of the deficit in olivary serotonin, the timing of the seizure, and the demonstration of the necessity of the inferior olive for myoclonus suggest that pathological inferior olivary activity contributes to the genesis of a myoclonic seizure.

Microinjection of L-glutamate into a subregion of the gigantocellular nucleus of the rat medulla oblongata significantly lowers arterial pressure. This vasodepressor area, the gigantocellular depressor area, is topographically distinct from other vasoactive areas of the medulla. We sought to determine the efferent projections of the gigantocellular depressor area and compare these to the efferent projections of sympathoexcitatory neurons within the rostral ventrolateral medulla. The anterograde tracer Phaseolus vulgaris-leucoagglutinin was deposited into sites in the gigantocellular depressor area or rostral ventrolateral medulla (pressor area) functionally defined as vasodepressor or vasopressor by microinjections of L-glutamate. Following Phaseolus vulgaris-leucoagglutinin injections into the gigantocellular depressor area, labeled punctuate fibers were seen bilaterally within distinct areas of a number of autonomic regions including the nuclei of the solitary tract, subcoeruleus area, parabrachial complex, the medial medullary reticular formation of the medulla and pons, and laminae 7 and 10 of the thoracic spinal cord. Following deposits into the rostral ventrolateral medulla (pressor area), labeled fibers were seen in many of these same autonomic nuclei; however, efferents from the gigantocellular depressor area to the nucleus of the solitary tract, the parabrachial complex and the reticular formation were medial to rostral ventrolateral medulla (pressor area) efferents to these same areas. These data indicate that neurons within the gigantocellular depressor area and the rostral ventrolateral medulla (pressor area) project to autonomic nuclei throughout the central nervous system and further suggest a heterogeneity of function with regard to autonomic control both within the reticular formation and its efferent targets. In addition, these data support the view that the gigantocellular depressor area may be a novel reticulospinal sympathoinhibitory area.