Distribution and developmental expression of octopamine‐immunoreactive neurons in the central nervous system of the leech

Abstract

Octopamine, a biogenic amine analogous to norepinephrine, plays an important role in the orchestration and modulation of invertebrate behavior. In the leech, the behavioral actions of octopamine have been demonstrated; however, identification of octopaminergic neurons had not been determined by using immunohistochemical techniques. Thus, we used an antibody highly specific to octopamine to examine the distribution of octopamine-immunoreactive neurons in the segmental ganglia of American and European medicinal leeches (Macrobdella decora and Hirudo medicinalis). One pair of octopamine-immunoreactive neurons was located in the dorsolateral ganglionic region of anterior ganglia 1-6 and posterior ganglia 15-21. No corresponding octopamine-immunoreactive neurons were found in midbody ganglia 7-14. Using Neutral Red staining in combination with intracellular Neurobiotin injections and octopamine immunostaining, we determined the identity of the dorsolateral octopamine-immunoreactive cells. The dorsolateral octopamine-immunoreactive neuron (the DLO) was not cell 21, the only previously reported Neutral Red staining neuron in the dorsolateral position. We also determined that the Leydig neuron was not octopamine immunoreactive in either of the two medicinal leech species. Octopamine immunostaining in the sex ganglia revealed hundreds of immunoreactive neurons in sexually mature leeches. Such neurons were not observed in juvenile leeches. The developmental time course of octopamine immunoreactivity in the dorsolateral octopamine-immunoreactive neurons was also investigated by staining embryonic Hirudo medicinalis. Octopamine expression occurred relatively late as compared with the detectable onset of serotonin expression. Octopamine expression in the dorsolateral octopamine-immunoreactive cells was not detectable at early to mid-embryonic stages, and must commence during late embryonic to early juvenile stages. The identification of octopamine-immunoreactive cells now sets the stage for further investigations into the functional role of octopamine in leech behavior and the development of behavior.

Dopamine Activates the Motor Pattern for Crawling in the Medicinal Leech

“…Cell identities were further confirmed by iontophoretically injecting 5% Neurobiotin (ϩ0.5-1 nA) into a given cell for at least 10 min. Tissue was subsequently fixed, processed, treated with Cy3-conjugated streptavidin (1:50 dilution in PBS for 18 -24 h at 4°C; Jackson ImmunoResearch, West Grove, PA), and prepared for viewing according to the methods of Gilchrist et al (1995). Dopamine superfusion.…”

Fine structure of the ventral nerve centre and interspecific identification of individual neurons in the enigmatic Chaetognatha

“…Within the Protostomia, individually identiWable neurons have been shown to be present in the nervous systems of, e.g. Arthropoda (Burrows 1996;Harzsch et al 2005;Harzsch 2006), Annelida (Stuart et al 1987;Huang et al 1998, Gilchrist et al 1995Brodfuehrer and Thorogood 2001;Orrhage and Müller 2005;Müller 2006), Nemathelminthes/Cycloneuralia (White et al 1986;Walthall 1995), basal Mollusca (Friedrich et al 2002;Voronezhskaya et al 2002), Plathelminthes (Halton and Gustafsson 1996;Reuter et al 1998;Reuter and Halton 2001), and Gnathifera (Müller and Sterrer 2004). The presence of at least some individually identiWable neurons in basal deuterostomes such as tunicates (Meinertzhagen 2004;Stach 2005;Meinertzhagen et al 2004;Imai and Meinertzhagen 2007;Soviknes et al 2007), and the lancelet (Wicht and Lacalli 2005) indicates that the potential to establish individual identities may not only be present in the ground pattern of Protostomia; but may even date back to the ground pattern of Bilateria.…”

Beyond the central pattern generator: amine modulation of decision-making neural pathways descending from the brain of the medicinal leech

“…Methods used for the 5-HT immunostaining of leech embryos have been described previously in detail (Gilchrist et al, 1995). Briefly, a goat anti-5-HT antiserum (gift from Dr Robert Elde, University of Minnesota) was used at a dilution of 1:200.…”

“…Following staining, all tissues were washed in hypo-osmotic Millonig's buffer (Gilchrist et al, 1995), dehydrated through a graded ethanol series, and specimens were mounted between coverslips in Depex mounting medium (Electron Microscopy Sciences, Fort Washington, PA, USA). An MRC 1024 Laser Scanning Confocal Microscope (Bio-Rad, Hercules, CA, USA) mounted on an AX70 microscope equipped for epifluorescence (Olympus, Lake Success, NY, USA) was used to view and image mounted samples as described (Mesce et al, 1993).…”

“…A laser scanning confocal micrograph of the leech brain is shown in Fig.·1. Using the same 5-HT antibody we reported on previously in segmental ganglia (Gilchrist et al, 1995), we immunolabeled a population of somata and their associated processes (Fig.·1). A dense network of 5-HTimmunoreactive (5-HT-ir) fibers was associated with the SEG and SPEG (N=6).…”