Cloning and functional characterization of a novel dopamine receptor from Drosophila melanogaster.

A cDNA clone is described that encodes a novel G-protein-coupled dopamine receptor (DopR99B) expressed in Drosophila heads. The DopR99B receptor maps to 99B3-5, close to the position of the octopamine/tyramine receptor gene at 99A10-B1, suggesting that the two may be related through a gene duplication. Agonist stimulation of DopR99B receptors expressed in Xenopus oocytes increased intracellular Ca2+ levels monitored as changes in an endogenous inward Ca2+-dependent chloride current. In addition to initiating this intracellular Ca2+ signal, stimulation of DopR99B increased cAMP levels. The rank order of potency of agonists in stimulating the chloride current is: dopamine > norepinephrine > epinephrine > tyramine. Octopamine and 5-hydroxytryptamine are not active (< 100 microM). This pharmacological profile plus the second-messenger coupling pattern suggest that the DopR99B receptor is a D1-like dopamine receptor. However, the hydrophobic core region of the DopR99B receptor shows almost equal amino acid sequence identity (40-48%) with vertebrate serotonergic, alpha 1- and beta-adrenergic, and D1-like and D2-like dopaminergic receptors. Thus, this Drosophila receptor defines a novel structural class of dopamine receptors. Because DopR99B is the second dopamine receptor cloned from Drosophila, this work establishes dopamine receptor diversity in a system amenable to genetic dissection.

The association of octopamine with specific neurones along lobster nerve trunks.

Octapamine and its synthetic enzyme, tyramine beta-hydroxylase (TBH), are found in high concentrations at two points along second thoracic nerve roots in lobsters. The first is in the proximal section of the second root between the ventral nerve cord and the bifurcation of the root into medial (to flexor muscles) and lateral (to extensors) branches. The second region of high concentration is within a well known crustacean neurosecretory system, the pericardial organ, located close to the ends of the lateral branches of the roots. 2. With several different staining procedures, small clusters of nerve cell bodies are found within the connective tissue sheath in the proximal regions of the second roots. No cell bodies are seen in the pericardial organ regions. Cell bodies are variable in number and position between corresponding roots in the same animal and homologous roots among different animals. The average numbers of cell bodies, however, correlate well with TBH and octopamine content, and with the synthesis of octopamine in these same regions of roots. 3. Small clusters of root cell bodies dissected from preparations have greater than 500-fold higher activities of TBH than isolated efferent excitatory and inhibitory or afferent sensory axons. 4. Along with octopamine, the preferential synthesis of acetylcholine and serotonin is also seen in proximal segments of roots. Acetylcholine synthesis in these regions may represent transmitter synthesized in the nerve terminals innervating the root cells. The role of serotonin in these regions is not understood at this time but the amounts of endogenous serotonin found are only a tenth of the amounts of octopamine present. 5. Dopamine is not synthesized from tyrosine in second thoracic roots. However, if DOPA or dopamine are used as precursor compounds, then noradrenaline, which is usually not found in lobsters, can be accumulated in proximal segments of roots. 6. Phenolamines are converted to two further metabolites by lobster tissues. The compounds are unidentified and are named fast and slow product on the basis of their migration on electrophoresis at acid pH. Some partial characterization of slow product reveals that it is a mixture of compounds that can be converted on mild acid hydrolysis to fast product and the parent phenolamine. 7. The several lines of evidence presented suggest that nerve cells found in the proximal segments of the second thoracic roots contain and can synthesize octopamine. Since not all the cells in any single root have been analysed for octopamine or TBH, however, the possibility that one or more of the cells contain physiologically interesting substances other than octopamine is not eliminated.

Octopamine release at two points along lobster nerve trunks.

Nerve cells in the proximal regions of second thoracic roots in lobsters have been injected with the fluorescent dye Procion Yellow. Examination of the roots reveals an elaborate array of cell branches in a superficial layer of the root in the vicinity of the cell bodies. Large varicosities, up to 10 mum in diameter, are seen lined up along fine nerve branches. 2. In these same regions, electron microscopic examination shows the presence of large profiles filled with 0-1-0-2 mum dense cored granules, and having the appearance of nerve endings. These profiles probably correspond to the varicosities seen in the Procion Yellow injections. The dense cored granules within the endings have a crystalline substructure. All the endings are found within 7 mum of the surface of the root and no obvious physiological target tissue exists in their surroundings. Endings have not been traced directly to root cell bodies.However, granules of similar dimensions to those seen in endings are found in cell bodies, axon-hillock regions and numerous axonal profiles in the superficial root regions near cell bodies. The morphological studies suggest that the root neurones have the typical appearance of neurosecretory cells. 3. Octopamine pools in cell body regions of second thoracic roots can be isotopically labelled by incubation with either [3H]tyramine or [3H]-tyrosine. After labelling, pulsing with 100 mM potassium causes an increase in the rate of release of radioactive material. Upon return to normal media background rates of release are re-established. The enhanced efflux has the following properties: (a) repeated pulses of potassium release less radio-active material each time; (b) a prolonged potassium pulse produces first a peak of release, then a decline to a plateau, and the plateau level of release is maintained for the duration of the potassium pulse; (c) release is dependent on the presence of calcium ions in the bathing fluid and 40 mM cobalt prevents release; (d) release is selective for octopamine. With tyrosine as a precursor compound, as much radioactive tyrosine as octopamine is found in tissues after incubation, yet pulsing with potassium causes an enhanced efflux only of octopamine from preparations. 4. Release of octopamine also can be demonstrated from pericardial organs near the ends of lateral branches of the roots and the properties of the release are identical to those seen with cell body regions. 5. Physiological studies, in which root cells are antidromically activated while recording from cell bodies, suggest that the distal endings of at least some of the root cells are at the pericardial organs. 6. The results suggest that root cell neurones are neurosecretory cells capable of releasing octopamine at two points: one near cell bodies, the other at the pericardial organs near the distal ends of the roots...

Octopamine neurons in lobsters: location, morphology, release of octopamine and possible physiological role.

Octopamine cells are found along second thoracic roots, where they serve as neurosecretory neurons capable of releasing octopamine at two distinct points: one into the hemolymph immediately before it enters the gills; one into the hemolymph immediately after it leaves the gills. The octopamine cells receive a cholinergic synaptic input. We presume that this input is from processes of peripheral sensory cells bringing information to the CNS. Octopamine can increase the strength of contraction of exoskeletal muscles and, at higher concentrations, can induce contractures in these muscles. These effects can be interpreted as a resetting of the level of ionized calcium within muscle fibers (the contracture) to a higher value or a possible enhanced entry of calcium ions during nerve stimulation (increased strength of contraction). The observed effects are of a prolonged duration, outlasting the time of application of octopamine by some 20-40 minutes. We do not know if this effect on muscle tension production is the normal physiological role of octopamine. Other possible roles will be explored in the future. The pathway involving the octopamine neurons in lobsters may provide a model neurohumoral system that can be studied and understood in detail from the level of sensory input to the level of behavioral output.