Summer Research

* Student(s): Matthew Ruggieri , Carlos Mercedes
Advisor: Herman Lehman

Title: Tyramine-β-Hydroxylase Activity as a Means of Localizing Octopamine in the Terminal Abdominal Ganglia of Manduca Sexta


Octopamine (OA), a biogenic amine, is an essential component of the invertebrate nervous system. The synthesis of OA from tyramine (TA) is catalyzed by tyramine-β-hydroxylase (TBH). In previous immunocytochemical studies, octopaminergic-immunoreactive (OA-ir) cells were observed along the midline of all abdominal ganglia, including the terminal abdominal ganglia (TAG) (Lehman et al.,2000). In addition, octopamine-ir was observed among cells in the peripheral regions of the TAG. In contrast, Kate Sanborn et al. have observed TBH mRNA in neurons only along the medial line. This study is focused on clarifying the location of OA neurons in the insect nervous system.

To clarify the location of octopaminergic neurons, we have measured TBH activity levels and octopamine and tyramine levels in microdissected regions of the terminal abdominal ganglia. Late pupal stage 18 Manduca sexta were dissected, the terminal abdominal ganglia were removed and sectioned into medial and lateral portions. A TBH assay (based on the conversion of [3H] tyramine to [3H] octopamine) was used to estimate the relative levels of TBH activity. Endogenous OA levels were measured on dilute perchloric acid extracts of medial and lateral regions of the TAG with High Performance Liquid Chromatography (HPLC) and electrochemical detection.

Three experimental runs comparing lateral and medial TBH activity yielded varying relative lateral/medial levels. The results of the first run indicated greater TBH activity in the lateral region. The second and third runs indicated however, that the lateral and medial regions had indistinguishable levels of TBH activity. Our experimental data are evidence that TBH enzymatic activity is not confined strictly to either the medial or lateral regions of the insect TAG. The direct OA measurements suggest that there is a small amount of octopamine produced in both the medial and lateral regions of the TAG.

The results we obtained support the possible explanation that dendritic arbors innervating the lateral region of the TAG allow for octopamine detection throughout the ganglion. Furthermore, analysis shows that there is no statistical difference between OA levels/TBH activity in the medial and lateral regions (Student t-test). Future work will revolve around the use of single ganglia, whose medial and lateral portions cannot easily be confused, thus avoiding possible heterogeneity in the sampling and dissection of the TAG.

Student stipend support for MR provided by the Ralph E. Hansmann Science Student Support Fund. Support for CM provided by a National Science Foundation Research Grant to Herman Lehman, in collaboration with Gene Robinson at the University of Illinois, Urbana-Champaign.