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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.

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 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 gigantocellular depressor area (GiDA) is a functionally defined subdivision of the medullary gigantocellular reticular formation where vasodepressor responses are evoked by glutamate microinjections (Aicher, S. A., D. J. Reis, D. A. Ruggiero, and T. A. Milner. Neuroscience 60: 761-779, 1994). The present experiments sought to determine whether the GiDA 1) tonically inhibits the sympathetic nervous system; 2) is necessary for baroreflex function; and 3) is functionally distinct from adjacent vasodepressor regions in the medullary reticular formation, including the midline raphe nuclei and the caudal ventrolateral medulla (CVL). Excitotoxic lesions of the GiDA abolished the baroreflex and significantly increased sympathetic nerve activity in anesthetized rats. Equivalent injections into the midline raphe nuclei elevated sympathetic activity but did not alter baroreflex responses. Therefore, the GiDA is functionally distinct from the raphe nuclei, although both contain tonically active sympathoinhibitory neurons. Because the effects of GiDA lesions were identical to those seen after lesions of the CVL, further studies were required to demonstrate that the GiDA and CVL are functionally and anatomically distinct. First, intramedullary injections of kynurenic acid produced hypertension and blocked the baroreflex when placed in the CVL, but not when placed in the GiDA. Second, muscimol inactivation of the RVL blocked the hypertension produced by excitotoxic lesions of the CVL, but failed to block the hypertension produced by similar lesions of the GiDA. Third, CVL neurons project to the RVL but not the spinal cord, whereas GiDA neurons project to the spinal cord but not the RVL. These studies show that the CVL and GiDA are both tonically sympathoinhibitory regions, but they are distinct with regard to their functional connectivity with other autonomic regions.

Microinjection of L-glutamate into a restricted area of the medullary gigantocellular reticular formation, the gigantocellular depressor area (GiDA), lowers arterial pressure. Unlike the nuclei tractus solitarii and the caudal ventrolateral medulla, the two principle medullary vasodepressor areas, the GiDA projects directly to the spinal cord and not to the rostral ventrolateral medulla (Aicher et al. [1994] Neuroscience 60:761-779). We investigated whether neurons within GiDA directly innervate autonomic areas of the thoracic spinal cord. Fluoro-Gold injected into the thoracic spinal cord labeled neurons within functionally defined vasodepressor sites in the GiDA in the same animal. To examine the morphology of GiDA efferents to the spinal cord, the anterograde tracer Phaseolus vulgaris-leucoagglutinin was iontophoresed into the GiDA, and efferent processes in the intermediolateral cell column and nucleus intercalatus spinalis were examined by electron microscopy. Labeling was confined to axons and axon terminals (n = 144) that usually contained primarily small clear vesicles, contacted large and small dendrites, and formed symmetric (inhibitory) synapses. To determine whether some of the postsynaptic targets of GiDA efferent terminals in the thoracic spinal cord were sympathoadrenal preganglionic neurons, these neurons were retrogradely labeled from the adrenal gland with Fluoro-Gold in rats that had deposits of the anterograde tracer, biotinylated dextran amine (BDA), in the GiDA. Some BDA-containing terminals formed symmetric synapses with dendrites containing Fluoro-Gold. We conclude that a population of neurons in the GiDA monosynaptically innervates some sympathetic preganglionic neurons. The findings suggest the presence of a novel reticulospinal sympathoinhibitory projection originating in the GiDA.

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.