Distribution and development of dopamine‐ and octopamine‐synthesizing neurons in the medicinal leech

Abstract

Although the medicinal leech is a well-studied system in which many neurons and circuits have been identified with precision, descriptions of the distributions of some of the major biogenic amines, such as dopamine (DA) and octopamine (OA), have yet to be completed. In the European medicinal leech Hirudo medicinalis and the American medicinal leech Macrobdella decora,we have presented the first immunohistochemical study of DA neurons in the entire central nervous system, and of OA-immunoreactive (ir) neurons in the head and tail brains. Dopaminergic neurons were identified using the glyoxylic acid method and antisera to DA and its rate-limiting synthetic enzyme tyrosine hydroxylase (TH). Octopaminergic neurons were recognized using a highly specific antiserum raised against OA. An antibody raised against DA-beta-hydroxylase (DbetaH), the mammalian enzyme that converts DA to norepinephrine (NE), was found to immunostain OA-ir neurons. This antibody appears to cross-react with the closely related invertebrate enzyme tyramine-beta-hydroxylase, which converts tyramine to OA, suggesting that the OA-ir cells are indeed octopaminergic, capable of synthesizing OA. Because the DbetaH antiserum selectively immunostained the OA-ir neurons, but not the DA-synthesizing cells, our results also indicate that the DA-ir neurons synthesize DA and not NE as their end product. The expression of TH immunoreactivity was found to emerge relatively early in development, on embryonic day 9 (47-48% of development). In contrast, OA expression remained absent as late as embryonic day 20. Higher order processes of some of the dopaminergic and octopaminergic neurons in the adult brain were observed to project to a region previously described as a neurohemal complex. Several TH-ir processes were also seen in the stomatogastric nerve ring, suggesting that DA may play a role in the regulation of biting behavior. By mapping the distributions and developmental expression pattern of DA and OA neurons in the leech, we aim to gain a better understanding of the functional roles of aminergic neurons and how they influence behavior.

A cephalic projection neuron involved in locomotion is dye coupled to the dopaminergic neural network in the medicinal leech

“…Tr2 and the dopaminergic system of the head brain (Fig.·1), a network of cells that was recently described by Crisp et al (2002). Examination of all head brains (N=7) in which cell Tr2 was filled with Neurobiotin revealed a collection of dye-coupled neurons in the SEG.…”

“…Tissues were incubated overnight at 4°C in a 1:100 dilution of streptavidin [conjugated to the cyanine fluorophore Cy3 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA)] in hypo-osmotic Millonig's buffer (13·mmol·l -1 NaH 2 HPO 4 , 86·mmol·l -1 Na 2 HPO 4 , pH·7.8). Labeling DA-synthesizing neurons in the leech CNS was conducted according to the methods of Crisp et al (2002). Briefly, ganglia (in which neurons had been filled with Neurobiotin) were incubated for 48·h in a mouse monoclonal antiserum raised against TH, the rate-limiting enzyme in the synthesis of DA, diluted to 1:100 in hypo-osmotic Millonig's buffer (containing 3% normal goat serum and 0.3% Triton X-100).…”

“…Briefly, ganglia (in which neurons had been filled with Neurobiotin) were incubated for 48·h in a mouse monoclonal antiserum raised against TH, the rate-limiting enzyme in the synthesis of DA, diluted to 1:100 in hypo-osmotic Millonig's buffer (containing 3% normal goat serum and 0.3% Triton X-100). This antibody was shown previously to stain selectively only leech neurons that expressed immunoreactivity to DA (Crisp et al, 2002). Tissues were then incubated for 48·h in a donkey anti-mouse antiserum conjugated to the cyanine fluorophore Cy5 (Jackson ImmunoResearch) diluted at 1:100 in hypo-osmotic Millonig's buffer (containing 3% normal goat serum and 0.3% Triton X-100).…”

“…Here, we describe coupling between the DA-synthesizing neurons of the leech head brain (Crisp et al, 2002) and the descending brain interneuron, Tr2, shown previously to terminate (O'Gara and Friesen, 1995;Taylor et al, 2003) or trigger (Brodfuehrer and Friesen, 1986) swimming. Our report is significant to the field of locomotion, because no other studies have previously documented the physiological actions of DA on the swim or crawl neural networks of the leech.…”

Dopamine Activates the Motor Pattern for Crawling in the Medicinal Leech

“…Based on data obtained in the single ganglion, we can conclude that the rhythm-generating circuitry for crawling exists within the smallest segmental division of the leech CNS, thus empirically defining the unit burst generator for crawling. In addition, because each body segment contains one unique pair of DA-containing neurons, which richly innervates each segmental ganglion or neuromere (Crisp et al, 2002;Crisp and Mesce, 2004), the release of DA via these neurons probably provides the natural route for the putative actions of DA on the crawl CPG.…”