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Cholecystokinin-tetrapeptide (CCK-4) induces panic attacks both in patients with panic disorder (PD) and healthy volunteers. It has been shown that panic elicited by CCK-4 is improved after treatment with antidepressants. Moreover, a reduction of CCK-4-induced panic has also been demonstrated after treatment with lorazepam in single subjects and after selective GABAergic treatment with vigabatrin. Although benzodiazepines are widely used as anxiolytics, no controlled study on the effects of benzodiazepines on CCK-4-induced panic symptoms is available so far. Therefore, we investigated the effects of alprazolam and placebo on CCK-4-induced panic symptoms in a double-blind, placebo-controlled study. A total of 30 healthy subjects were challenged with 50 µg CCK-4. Out of these 30 subjects, 26 showed a marked panic response to CCK-4. Subjects were rechallenged after a 7-day interval and treated with 1 mg alprazolam or placebo 1 h prior to the second CCK-4 challenge. Panic was assessed using the acute panic inventory (API) and a DSM-IV-derived panic symptom scale (PSS). Moreover, the number of reported symptoms and self-rated anxiety and arousal were recorded. We found a significant reduction of the API and PSS scores and of the number of reported symptoms compared to placebo. Moreover, compared to placebo the CCK-4-induced ACTH and cortisol release were significantly attenuated during the CCK-4 challenge after alprazolam treatment. However, also placebo treatment reduced CCK-4-induced anxiety and HPA-axis activation to a certain extent. In conclusion, our data show that alprazolam reduces CCK-4-induced panic, which supports the hypothesis of a possible interaction between the GABA and the CCK system. [ABSTRACT FROM AUTHOR]

Competing Interests: Frank G. Rücker reports honoraria from and consultancy for Jazz Pharmaceuticals, Novartis, and BMS/Celgene; travel support from Jazz Pharmaceuticals. Michael Lübbert reports an advisory role for Abbvie, Astex Pharmaceuticals, Imago BioSciences, Janssen, Otsuka, and Syros; research support from Janssen and Cheplapharm. Michael W. M. Kühn reports honoraria from and consultancy for Pfizer, Kura Oncology, Jazz Pharmaceuticals, BMS/Celgene, and Abbvie; speakers bureau of Gilead. Lars Bullinger reports honoraria from Abbvie, Amgen, Astellas, BMS/Celgene, Daiichi Sankyo, Gilead, Janssen, Jazz Pharmaceuticals, Menarini, Novartis, Pfizer, Roche, and Sanofi; research support from Bayer and Jazz Pharmaceuticals. Verena I. Gaidzik reports an advisory role for Jazz Pharmaceuticals, Abbvie, and Boehringer‐Ingelheim; speakers bureau of Pfizer, Janssen, and Abbvie; and travel support from Abbvie. Frank Stegelmann reports honoraria from and consultancy for AOP Pharma, MorphoSys, BMS/Celgene, Incyte, Novartis, and Pfizer. Felicitas Thol reports an advisory role for Novartis, BMS, Abbvie, Menarini, and Rigel. Michael Heuser reports honoraria from Certara, Jazz Pharmaceuticals, Janssen, Novartis, and Sobi; paid consultancy for Abbvie, Amgen, BMS/Celgene, Glycostem, LabDelbert, Pfizer, PinotBio, and Servier; and research funding to his institution from Abbvie, Agios, Astellas, BMS/Celgene, Glycostem, Jazz Pharmaceuticals, Karyopharm, Loxo Oncology, and PinotBio. Hartmut Döhner declares being in an advisory role for Abbvie, Agios, Amgen, Astellas, AstraZeneca, Berlin Chemie, BMS/Celgene, Daiichi Sankyo, GEMoaB, Gilead, Janssen, Jazz Pharmaceuticals, Novartis, Servier, Stemline, and Syndax; research funding from Abbvie, Agios, Amgen, Astellas, BMS/Celgene, Jazz Pharmaceuticals, Kronos Bio, Novartis, and Pfizer. Konstanze Döhner reports an advisory role for Amgen, BMS/Celgene, Daiichi Sankyo, Janssen, Jazz Pharmaceuticals, Novartis, and Roche; research funding from Agios, Astex, Astellas, BMS/Celgene, and Novartis. All other authors declare no competing interest. The remaining authors declared no conflicts of interest.

Glutamate is the most important excitatory neurotransmitter in the brain. The ability to assess glutamate release and re-uptake with high spatial and temporal resolution is crucial to understand the involvement of this primary excitatory neurotransmitter in both normal brain function and different neurological disorders. Real-time imaging of glutamate transients by fluorescent nanosensors has been accomplished in rat brain slices. We performed for the first time single-wavelength glutamate nanosensor imaging in human cortical brain slices obtained from patients who underwent epilepsy surgery. The glutamate fluorescence nanosensor signals of the electrically stimulated human cortical brain slices showed steep intensity increase followed by an exponential decrease. The spatial distribution and the time course of the signal were in good agreement with the position of the stimulation electrode and the dynamics of the electrical stimulation, respectively. Pharmacological manipulation of glutamate release and reuptake was associated with corresponding changes in the glutamate fluorescence nanosensor signals. We demonstrated that the recently developed fluorescent nanosensors for glutamate allow to detect neuronal activity in acute human cortical brain slices with high spatiotemporal precision. Future application to tissue samples from different pathologies may provide new insights into pathophysiology without the limitations of an animal model., (© 2022. The Author(s).)

The protozoan parasite Trypanosoma brucei is the causative agent of human African trypanosomiasis (HAT). The disease is fatal if it remains untreated, whereas most drug treatments are inadequate due to high toxicity, difficulties in administration, and low central nervous system penetration. T. brucei glycogen synthase kinase 3 short ( Tb GSK3s) is essential for parasite survival and thus represents a potential drug target that could be exploited for HAT treatment. Indirubins, effective leishmanicidals, provide a versatile scaffold for the development of potent GSK3 inhibitors. Herein, we report on the screening of 69 indirubin analogues against T. brucei bloodstream forms. Of these, 32 compounds had potent antitrypanosomal activity (half-maximal effective concentration = 0.050 to 3.2 μM) and good selectivity for the analogues over human HepG2 cells (range, 7.4- to over 641-fold). The majority of analogues were potent inhibitors of Tb GSK3s, and correlation studies for an indirubin subset, namely, the 6-bromosubstituted 3'-oxime bearing an extra bulky substituent on the 3' oxime [(6-BIO-3'-bulky)-substituted indirubins], revealed a positive correlation between kinase inhibition and antitrypanosomal activity. Insights into this indirubin- Tb GSK3s interaction were provided by structure-activity relationship studies. Comparison between 6-BIO-3'-bulky-substituted indirubin-treated parasites and parasites silenced for Tb GSK3s by RNA interference suggested that the above-described compounds may target Tb GSK3s in vivo To further understand the molecular basis of the growth arrest brought about by the inhibition or ablation of Tb GSK3s, we investigated the intracellular localization of Tb GSK3s. Tb GSK3s was present in cytoskeletal structures, including the flagellum and basal body area. Overall, these results give insights into the mode of action of 6-BIO-3'-bulky-substituted indirubins that are promising hits for antitrypanosomal drug discovery., (Copyright © 2019 American Society for Microbiology.)

Clinical studies have suggested association of some hepatitis C virus (HCV) subtypes or isolates with progression toward hepatocellular carcinoma (HCC). HCV core protein has been reported to interfere with host Wnt/β-catenin pathway, a cell fate-determining pathway, which plays a major role in HCC. Here, we investigated the impact of HCV core genetic variability in the dysregulation of Wnt/β-catenin pathway. We used both transient expression of core proteins from clinical isolates of HCV subtypes 1a (Cambodia), 4a (Romania) and 4f (Cameroon) and infection systems based on a set of engineered intergenotypic recombinant viruses encoding core from these various clinical strains. We found that TCF transcription factor-dependent reporter activity was upregulated by core in a strain-specific manner. We documented core sequence-specific transcriptional upregulation of several β-catenin downstream target genes associated with cell proliferation and malignant transformation, fibrogenesis or fat accumulation. The extent of β-catenin nuclear translocation varied in accordance with β-catenin downstream gene upregulation in infected cells. Pairwise comparisons of subgenotypic core recombinants and mutated core variants unveiled the critical role of core residues 64 and 71 in these dysregulations. In conclusion, this work identified natural core polymorphisms involved in HCV strain-specific activation of Wnt/β-catenin pathway in relevant infection systems.

The reaction of the dimer [Rh(III)(pentamethylcyclopentadienyl)(μ-Cl)Cl]2 ([Rh(III)(Cp*)(μ-Cl)Cl]2) with the hydroxypyrone ligands maltol and allomaltol affords complexes of the general formula [Rh(III)(Cp*)(L)Cl] under standard and microwave conditions. The organometallic compounds were characterized by standard analytical methods and in the case of the allomaltol derivative in the solid state by single-crystal X-ray diffraction analysis. The complexes showed similar cytotoxicity profiles and were proved to be moderately active against various human cancer cell lines. The stoichiometry and stability of these complexes were determined in aqueous solution by pH-potentiometry, (1)H NMR spectroscopy and UV-visible spectrophotometry. Speciation was studied in the presence and in the absence of chloride ions. Hydrolysis of [Rh(III)(Cp*)(H2O)3](2+) gave dimeric mixed hydroxido species [(Rh(III)(Cp*))2(μ-OH)3](+) and [(Rh(III)(Cp*))2(μ-OH)2Z2] (Z=H2O/Cl(-)). Formation of the mononuclear complexes [Rh(III)(Cp*)(L)Z] of maltol and allomaltol with similar and moderate stability was found. These species predominate at physiological pH and decompose only partially at micromolar concentrations. In addition, hydrolysis of the aqua complex or a chlorido/hydroxido co-ligand exchange resulted in the formation of the mixed-hydroxido species [Rh(III)(Cp*)(L)(OH)] in the basic pH range. Replacement of the chlorido by an aqua ligand in the complex [Rh(III)(Cp*)(L)Cl] was monitored and with the help of the equilibrium constants the extent of aquation at various chloride concentrations of the extra- and intracellular milieu can be predicted. Complexation of these Rh(III) complexes was compared to analogous [Ru(II)(η(6)-p-cymene)] species and higher conditional stabilities were found in the case of the Rh(III) compounds at pH7.4., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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)

1. The present article reviews several aspects of opioid signalling in the rostral ventrolateral medulla (RVLM) and their implications for the neural control of blood pressure. 2. In the RVLM, preproenkephalin (PPE) mRNA is expressed by bulbospinal cells that are strongly barosensitive. These putative presympathetic neurons includes C1 and non-C1 neurons. 3. In the RVLM, PPE mRNA is also present in GABAergic neurons that do not project to the thoracic spinal cord. 4. Rostral ventrolateral medulla presympathetic cells receive enkephalinergic inputs and express mu-opioid receptors (MOR). Some of their synaptic inputs also contain MOR. 5. Pre- and post-synaptic modulation of RVLM presympathetic neurons by MOR agonists has been demonstrated in slices of neonate brain. The post-synaptic effect is inhibitory (increased gK). Presynaptic effects include disfacilitation (reduction of glutamate release) and possibly dishinhibition (reduction of GABA release). 6. In conclusion, opioid signalling plays a pervasive role in the medullospinal network that controls sympathetic tone and arterial pressure. Opioid peptides are made by the presympathetic, presumably excitatory, cells of the RVLM and by local GABAergic inhibitory neurons. In addition, RVLM presympathetic neurons are also controlled by opioid peptides at the pre- and post-synaptic level. mu-Opioid receptors are found post-synaptically, whereas presynaptic receptors probably include both mu and delta subtypes. Conditions that trigger the release of opioid peptides by presympathetic neurons or by inputs to these cells are not fully understood and may include decompensated haemorrhage and certain types of peripheral sensory stimulation related to acupuncture.

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.

Physiological studies have suggested that mu-opioid receptor (MOR) activation can both excite and inhibit reticulospinal neurons in the rostral ventrolateral medulla (RVL), possibly via influences on GABAergic neurons. Thus, to determine the cellular relationships of MORs to GABAergic neurons in the RVL, two experimental approaches were used. First, single sections through the RVL were labeled for MOR using immunoperoxidase detection and for GABA using immunogold detection and examined by electron microscopy. These studies revealed that MOR-immunoreactive (IR) terminals were smaller on average than GABA-IR terminals and formed both asymmetric and symmetric synapses, whereas GABA-IR terminals formed exclusively symmetric synapses. MOR and GABA immunoreactivities rarely co-localized. Interactions between axons and terminals containing MOR or GABA immunoreactivity were primarily: (1) direct appositions with each other; or (2) convergence onto a common dendritic target that sometimes contained either MOR or GABA immunoreactivity. Since the identity of these target dendrites mostly was unknown, a second study was designed to determine if they might be reticulospinal neurons. For this study, reticulospinal neurons were identified with a retrograde tracer and both MOR and GABA were localized in the same sections of the RVL. These studies revealed that numerous GABA-IR terminals formed symmetric synapses on the perikarya and proximal dendrites of reticulospinal neurons. In contrast, few MOR-IR terminals contacted reticulospinal perikarya and large dendrites although they were often found nearby. These results provide anatomical evidence that MOR activation by endogenous or exogenous agonists may indirectly alter GABAergic neurotransmission in the RVL either through presynaptic interactions between cells or through competing influences on postsynaptic targets.

The central nervous system components for baroreflex regulation of sympathetic outflow include specific sets of neurons in the brain and spinal cord. Critical nuclei containing sympathetic baroreceptive neurons are the nuclei of the solitary tract, regions of the caudal and rostral ventrolateral medulla, and the intermediolateral cell column in the spinal cord. While many other brain regions project to these nuclei, cells in these areas appear to form the minimal required pathway for baroreflex control of sympathetic outflow. Synaptic connections have been identified between cells in these nuclei that are consistent with a serial relay from baroreceptor afferents through the brain stem and to sympathetic preganglionic neurons in the spinal cord. In recent years, we have examined the distribution of receptor proteins in these neurons, with a focus on receptors that are most likely to modulate the activity of these cells. In three studies examining the distribution of different receptors on distinct neurons, each study found some type of heterogeneity in the distribution of each receptor within a particular type of neuron. This heterogeneity was seen with regard to the distribution of receptor protein within the dendritic tree of individual neurons, as well as between pre- and postsynaptic sites on the same cell. This heterogeneous distribution of receptors suggests that receptors undergo dendritic targeting within autonomic neurons. This receptor trafficking may be regulated by heterogeneous afferent input to autonomic neurons and could be changed under conditions where afferent activity is significantly altered.

alpha-2A-adrenergic receptor (alpha2A-AR) agonists modulate diverse autonomic functions. These actions are believed to involve functionally specialized, second-order neurons in catecholamine-containing portions of the medial nucleus tractus solitarius (mNTS) at both intermediate (NTSi) and caudal (NTSc) levels. However, the cellular mechanisms subserving alpha2A-AR-mediated actions within the mNTS have yet to be established. Immunocytochemistry was employed to examine the subcellular distribution of alpha2A-AR in both the intermediate and caudal mNTS and its association with cells containing the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Quantitative regional comparison using immunogold showed that this receptor was distributed differentially to dendrites (NTSi, 46%; NTSc, 31%) and glia (NTSi, 29%; NTSc, 48%) at different levels of the NTS. Somata, axons, and terminals less frequently contained alpha2A-AR. The subcellular distribution of alpha2A-AR relative to catecholaminergic neurons also was similar within both subregions. Approximately 50% of alpha2A-AR-labeled somata also contained TH. In somatic profiles, alpha2A-AR labeling was often found in the cytosol and in association with endoplasmic reticulum and Golgi complexes, sites of receptor synthesis and trafficking. Approximately 20% of alpha2A-AR-immunoreactive dendrites also contained TH, where the receptor was often found on extrasynaptic portions of the plasma membrane near unlabeled terminals, some of which made symmetric contacts. However, TH-labeled terminals and dendrites usually were detected in the neuropil at a short distance (<10 microm) from alpha2A-AR-labeled neurons. alpha2A-AR-labeled glia frequently apposed unlabeled dendrites and terminals and were often located near TH-immunoreactive dendrites. These results indicate that, within the mNTS, alpha2A-AR is involved in a variety of autonomic processes, including postsynaptic modulation of mostly noncatecholaminergic dendrites, as well as influencing glia functions., (Copyright 2001 Wiley-Liss, Inc.)

Agonists of the mu-opioid receptor (MOR) have profound effects on blood pressure, heart rate, and respiration that may be mediated by C1 adrenergic neurons in the rostral ventrolateral medulla (RVL). C1 neurons are sympathoexcitatory and are involved in both tonic and reflex regulation of sympathetic outflow. This study was designed to determine whether C1 neurons, or their afferents, contain MOR. C1 neurons were identified by using an antibody against the epinephrine synthesizing enzyme phenylethanolamine-N-methyl transferase (PNMT), whereas MOR was localized by using an antipeptide antibody that recognizes the cloned MOR, MOR1. Combined immunoperoxidase and immunogold methods were used to examine the cellular distribution of MOR1 relative to PNMT-containing neurons in the RVL. MOR1 was found in 22% of PNMT-containing dendrites (n = 392), whereas MOR1-containing axons or axon terminals contacted 14% of PNMT-containing dendrites. This distribution was heterogenous with regard to dendritic size: PNMT-labeled dendrites containing MOR1 were usually large (60% were >1.2 microm), whereas PNMT-containing dendrites that received MOR1-labeled afferents were usually small (79% were <1.2 microm). Individual dendrites rarely contained MOR1 at both pre- and postsynaptic sites. Together these results suggest that MOR agonists may directly influence the activity of C1 neurons, as well as the activity of select afferents to these cells. Plasmalemmal membrane labeling for MOR1 was more frequent in smaller PNMT-containing dendrites, suggesting that postsynaptic receptors are more readily available for ligand binding in small dendrites, although the receptor was more frequently detected in larger PNMT dendrites. The selective distribution of MORs to specific pre- and postsynaptic sites suggests the receptor may be selectively trafficked to positions where it may regulate afferent activity that is heterogeneously distributed along the dendritic tree of C1 neurons., (Copyright 2001 Wiley-Liss, Inc.)

Agonists of the mu-opioid receptor (MOR) produce profound hypotension and sympathoinhibition when microinjected into the rostral ventrolateral medulla (RVL). These effects are likely to be mediated by the inhibition of adrenergic and other presympathetic vasomotor neurons located in the RVL. The present ultrastructural studies were designed to determine whether these vasomotor neurons, or their afferents, contain MORs. RVL bulbospinal barosensitive neurons were recorded in anesthetized rats and filled individually with biotinamide by using a juxtacellular labeling method. Biotinamide was visualized by using a peroxidase method and MOR was identified by using immunogold localization of an antipeptide antibody that recognizes the cloned MOR, MOR1. The subcellular relationship of MOR1 to RVL neurons with fast- or slow-conducting spinal axons was examined by electron microscopy. Fast- and slow-conducting cells were not morphologically distinguishable. Immunogold-labeling for MOR1 was found in all RVL bulbospinal barosensitive neurons examined (9 of 9). MOR1 was present in 52% of the dendrites from both types of cells and in approximately half of these dendrites the MOR1 was at nonsynaptic plasmalemmal sites. A smaller portion of biotinamide-labeled dendrites (16%) from both types of cells were contacted by MOR1-containing axons or axon terminals. Together, these results suggest that MOR agonists can directly influence the activity of all types of RVL sympathoexcitatory neurons and that MOR agonists may also influence the activity of afferent inputs to these cells. The heterogenous distribution of MORs within individual RVL neurons indicates that the receptor is selectively targeted to specific pre- and postsynaptic sites., (Copyright 2001 Wiley-Liss, Inc.)

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.

Ligands of the mu-opiate receptor (MOR) are known to influence many functions that involve vagal afferent input to the nucleus tractus solitarius (NTS), including cardiopulmonary responses, gastrointestinal activity, and cortical arousal. The current study sought to determine whether a cellular substrate exists for direct modulation of vagal afferents and/or their neuronal targets in the NTS by ligands of the MOR. Anterograde tracing of vagal afferents arising from the nodose ganglion was achieved with biotinylated dextran amine (BDA), and the MOR was detected by using antipeptide MOR antiserum. The medial subdivision of the intermediate NTS was examined by electron microscopy for the presence of peroxidase-labeled, BDA-containing vagal afferents and immunogold MOR labeling. MOR was present in both presynaptic axon terminals and at postsynaptic sites, primarily dendrites. In dendrites, MOR immunogold particles usually were located along extrasynaptic portions of the plasma membrane. Of 173 observed BDA-labeled vagal afferent axon terminals, 33% contained immunogold labeling for MOR within the axon terminal. Many of these BDA-labeled terminals formed asymmetric, excitatory-type synapses with dendrites, some of which contained MOR immunogold labeling. MORs were present in 19% of the dendrites contacted by BDA-labeled terminals but were present rarely in both the vagal afferent and its dendritic target. Together, these results suggest that MOR ligands modulate either the presynaptic release from or the postsynaptic responses to largely separate populations of vagal afferents in the intermediate NTS. These results provide a cellular substrate for direct actions of MOR ligands on primary visceral afferents and their second-order neuronal targets in NTS., (Copyright 2000 Wiley-Liss, Inc.)

Substance P (SP) is a peptide that is present in unmyelinated primary afferents to the dorsal horn and is released in response to painful or noxious stimuli. Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through modulation of responses to SP. MOR ligands may either inhibit the release of SP or reduce the excitatory responses of second-order neurons to SP. We examined potential functional sites for interactions between SP and MOR with dual electron microscopic immmunocytochemical localization of the SP receptor (NK1) and MOR in rat trigeminal dorsal horn. We also examined the relationship between SP-containing profiles and NK1-bearing profiles. We found that 56% of SP-immunoreactive terminals contact NK1 dendrites, whereas 34% of NK1-immunoreactive dendrites receive SP afferents. This result indicates that there is not a significant mismatch between sites of SP release and available NK1 receptors, although receptive neurons may contain receptors at sites distant from the peptide release site. With regard to opioid receptors, we found that many MOR-immunoreactive dendrites also contain NK1 (32%), whereas a smaller proportion of NK1-immunoreactive dendrites contain MOR (17%). Few NK1 dendrites (2%) were contacted by MOR-immunoreactive afferents. These results provide the first direct evidence that MORs are on the same neurons as NK1 receptors, suggesting that MOR ligands directly modulate SP-induced nociceptive responses primarily at postsynaptic sites, rather than through inhibition of SP release from primary afferents. This colocalization of NK1 and MORs has significant implications for the development of pain therapies targeted at these nociceptive neurons.

The ventrolateral portion of the periaqueductal gray (PAG) is one brain region in which ligands of the mu-opioid receptor (MOR) produce analgesia. In the PAG, MOR ligands are thought to act primarily on inhibitory [e.g., gamma-aminobutyric acidergic (GABAergic)] neurons to disinhibit PAG output rather than directly on medullary-projecting PAG neurons. In this study, the ultrastructural localization of MOR immunolabeling was examined with respect to either GABAergic PAG neurons or PAG projection neurons that were labeled retrogradely from the rostral ventromedial medulla. Immunoreactivity for MOR and GABA often coexisted within dendrites. Dual-labeled profiles accounted for subpopulations of dendrites containing immunoreactivity for either MOR (65 of 145 dendrites; 45%) or GABA (65 of 183 dendrites; 35%). In addition, nearly half of PAG neuronal profiles (148 of 344 profiles) that were labeled retrogradely from the ventromedial medulla contained MOR immunoreactivity. MOR was distributed equally among retrogradely labeled neuronal profiles in the lateral and ventrolateral columns of the caudal PAG. With respect to the presynaptic distribution of MOR, approximately half of MOR-immunolabeled axon terminals (35 of 69 terminals) also contained GABA. Some MOR and GABA dual-immunolabeled axon terminals contacted unlabeled dendrites (11 of 35 terminals), whereas others contacted GABA-immunoreactive dendrites (15 of 35 terminals). Furthermore, axon terminals synapsing on medullary-projecting PAG neurons sometimes contained immunoreactivity for MOR. These data support the model that MOR ligands can act by inhibiting GABAergic neurons, but they also provide evidence that MOR ligands may act directly on PAG output neurons. In addition, MOR at presynaptic sites could affect both GABAergic neurons and output neurons. Thus, the disinhibitory model represents only partially the potential mechanisms by which MOR ligands can modulate output of the PAG., (Copyright 2000 Wiley-Liss, Inc.)

Kappa opioid receptors (KORs) were immunocytochemically localized in the lumbosacral spinal cord of female rats in different stages of the estrous cycle to examine the influence of hormonal status on receptor density. KOR labeling was primarily in fine processes and a few neuronal cell bodies in the superficial dorsal horn and the dorsolateral funiculus. Quantitative light microscopic densitometry of the superficial dorsal horn revealed that rats in diestrus had significantly lower KOR densities than those in proestrus or estrus. This suggests that female reproductive hormones regulate spinal KOR levels, which may contribute to variations in analgesic effectiveness of KOR agonists across the estrous cycle.

Opiates active at the mu-opiate receptor (MOR) produce antinociception, in part, through actions involving substance P (SP), a peptide present in both unmyelinated primary afferents and interneurons within the dorsal horn. We examined potential functional sites for interactions between SP and MOR by using dual electron microscopic immunocytochemical localization of antisera against SP and a sequence-specific antipeptide antibody against MOR in rat cervical spinal dorsal horn. The distribution was compared with that of the functionally analogous dorsal horn of the trigeminal nucleus caudalis. Many of the SP-immunoreactive terminals in the dorsal horn contacted dendrites that contain MOR (53% in trigeminal; 70% in cervical spinal cord). Conversely, within the cervical spinal dorsal horn 79% of the MOR-labeled dendrites that received any afferent input were contacted by at least one SP-containing axon or terminal. Although SP-immunoreactive dendrites were rare, many of these (48%) contained MOR, suggesting that the activity of SP-containing spinal interneurons may be regulated by MOR ligands. A few SP-labeled terminals also contained MOR (12% in trigeminal; 6% in cervical spinal cord). These data support the idea that MOR ligands produce antinociception primarily through modulation of postsynaptic second-order nociceptive neurons in the dorsal horns of spinal cord and spinal trigeminal nuclei, some of which contain SP. They also suggest, however, that in each region, MOR agonists can act presynaptically to control the release of SP and/or glutamate from afferent terminals. The post- and presynaptic MOR sites are likely to account for the potency of MOR agonists as analgesics., (Copyright 2000 Wiley-Liss, Inc.)

Adrenergic (C1) neurons in the rostral ventrolateral medulla (RVL) are sympathoexcitatory and project directly to sympathetic preganglionic neurons (SPNs) in the thoracic spinal cord. C1 neurons also contain glutamate which may mediate the excitatory effects of RVL stimulation on SPNs through the N-methyl-D-aspartate (NMDA)-type receptor. Dual-labeling immunocytochemistry, combined with electron microscopy, was used to determine if NMDA receptors are located post-synaptic to adrenergic terminals in the spinal cord. Adrenergic terminals were labeled using an antibody directed against phenylethanolamine-N-methyl transferase (PNMT) and the NMDA receptor was identified using an antibody directed against the R1 subunit of the receptor (NMDAR1). NMDAR1 was found primarily in large and small dendrites and a few perikarya. The presence of NMDAR1 in the dendritic targets of PNMT-containing terminals was quantified in spinal cords sectioned either horizontally or coronally. PNMT-labeled terminals formed asymmetric synapses on dendrites containing immunogold labeling for NMDAR1, but NMDAR1 was more often detected in the targets of PNMT terminals when spinal cords were sectioned horizontally (59%) rather than coronally (28%). This difference in prevalence of NMDAR1 in targets of PNMT terminals is likely due to the preferential orientation of SPN dendrites in the horizontal plane, since longer dendritic shafts were visible in horizontal sections. When NMDAR1 was present in the dendritic targets of many adrenergic terminals, it was usually located at sites distal to the adrenergic input. We conclude that NMDA receptor ligands are likely to modulate the activity of dendritic targets of adrenergic terminals in the spinal cord, but are not closely associated with adrenergic synaptic input.

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.

The gigantocellular depressor area (GiDA) is a functionally defined subdivision of the medullary gigantocellular reticular formation where vasodepressor responses are evoked by glutamate nanoinjections. The GiDA also contains reticulospinal neurons that contain the alpha2A-adrenergic receptor (alpha2A-AR). In the present study, we sought to determine whether nanoinjections of the alpha2-AR agonist clonidine into the GiDA evoke cardiovascular responses and whether these responses can be attributed to the alpha2-AR. We found that nanoinjections of clonidine into the GiDA evoke dose-dependent decreases in arterial pressure and heart rate. These responses were equivalent in magnitude to responses produced by clonidine nanoinjections into the sympathoexcitatory region of the rostral ventrolateral medulla. Furthermore, the vasodepressor and bradycardic responses produced by clonidine injections into the GiDA were blocked in a dose-dependent fashion by the highly selective alpha2-AR antagonist 2-methoxyidazoxan, but not by prazosin, which is an antagonist at both the alpha1-AR and the 2B subtype of the alpha-AR. The antagonism by 2-methoxyidazoxan was site specific because injections of the antagonist into the rostral ventrolateral medulla failed to block the responses evoked by clonidine injections into the GiDA. These findings support the notion that clonidine produces sympathoinhibition through multiple sites within the medullary reticular formation, which is consistent with the wide distribution of the alpha2A-AR in reticulospinal neurons. These data also suggest that clonidine may have multiple mechanisms of action because it evokes a cardiovascular depressive response from regions containing neurons that have been determined to be both sympathoinhibitory and sympathoexcitatory.

The 2A subtype of the alpha-adrenergic receptor (alpha2A-AR) is necessary for the hypotensive effects of clonidine and other sympathoinhibitory adrenergic agonists. This hypotensive response appears to be due to the inhibition of sympathoexcitatory reticulospinal neurons found in the rostral ventrolateral medulla (RVL), including neurons of the C1 adrenergic cell group. The cellular mechanisms underlying this inhibition have not been established. Thus, this study examined the ultrastructural relationships between profiles containing alpha2AAR-immunoreactivity (alpha2AAR-I) and those containing the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) to determine potential cellular substrates for alpha2A-AR inhibition of C1 neuron activity. Consistent with previous light microscopic studies, alpha2AAR-I was found in perikarya and large dendrites and the majority of these profiles also contained TH-labeling (approximately 70% of 140). However, alpha2AAR-I in these cells was primarily found within endosomes and Golgi complexes and in clusters associated with the endoplasmic reticula, probable sites for synthesis and/or trafficking of receptors. In contrast, most of the alpha2AAR-I profiles (n=646) in the RVL were axons and axon terminals (approximately 68%) which lacked TH immunoreactivity. alpha2AAR-labeled axons were small and unmyelinated and labeled terminals usually formed symmetric synapses on the shafts of catecholaminergic or unlabeled dendrites. Most of these alpha2AAR-labeled axons were found in close proximity to TH-labeled profiles and approximately one-fifth (17% of 408) of the alpha2AAR-labeled axons and axon terminals directly contacted TH-labeled profiles, mostly dendrites. These studies suggest that alpha2AARs in the C1 area of the RVL function primarily as heteroreceptors on presynaptic axons and terminals of non-catecholaminergic cells, some of which provide inhibitory synaptic input to C1 neurons. These receptors may be activated by catecholamines released either from the dendrites of C1 neurons or from the terminals of other catecholaminergic neurons via volume transmission., (Copyright 1999 Elsevier Science B.V.)