Summer Research

* Student(s): Heather Shapiro , Katherine Hankowski
Advisor: Herman Lehman

Title: Localization of TβH in the CNS of Manduca sexta


The central nervous system of animals is composed of the brain and the nerve cord, while the rest of the nervous system makes up the peripheral nervous system. The neuron is the fundamental unit of both divisions of the nervous system, consisting of the axon, dendrite and cell body. Cell bodies are organized together to make ganglia. Neurons send, receive and integrate vast amounts of information that make animals aware of their surroundings.

Previous research has shown the importance of octopamine (OA) in the insect central nervous system. Lehman et al (2000) showed that steroid hormone is necessary and sufficient for adult expression of octopaminergic neurons in the abdominal ganglia. Lehman et al also showed that the OA immunoreactivity is localized in midline and lateral neurons of the adult terminal ganglia. Tyramine-β-hydroxylase (TβH) is the enzyme that converts tyramine to OA, and Kate Sanborn (2003) revealed that TβH mRNA appears to be located solely in midline neurons of larval terminal ganglia. Our research this summer focused on understanding the developmental regulation of TβH in all portions of the nervous system at different stages of development.

In Situ hybridization is a technique that uses a RNA or DNA probe to visualize mRNA in the cell. TβH mRNA was localized using a probe of RNA that was made to complement the TβH mRNA strand. The plasmids for the probe were isolated using a QIAgen midiprep kit, and the probe was bound using a 7-day protocol. Once the probe was bound, digoxigenin (DIG), an antigen in the RNA probe, was detected and amplified with an antibody conjugated to alkaline phosphatase (AP). The AP reacted with nitro-blue tetrazolium (NBT) in solution to create a dark stain sufficient to visualize the cells through a microscope.

Visualization of cells containing TβH mRNA revealed neurons in all regions of the insect nervous system. Dissecting the three stages of Manduca sexta: larval (5th), pupal (stage 14), and adult form (stage 18), allowed us to look at how the cells containing TβH change throughout development. There was high variation in the cells detected. In the abdominal ganglia of fifth larval instars, we observed one ganglion with one cells, 79 ganglia with two cells, five with three cells, and one ganglion with four. We observed this variation in all ganglia. In addition, the TβH mRNA-containing cells were detected in all regions of the nervous system of the fifth larval instar stage. In pupal and adult insects, we observed these cells in the abdominal region. In older insects (pupal; stages 14 ? 18) there was a notable increase in the color intensity of stained cells when compared to those of larval insects (5ths).

Our results suggest that TβH mRNA is abundant in the developing nervous system. Variability in the number of neurons, however, requires the development of additional methods (e.g. fluorescence and paraffin sectioning) to visualize all neurons in multiple planes of the ganglia. In addition, the increase in TβH mRNA intensity suggests that all OA neurons may be influenced by steroid hormone during development.

Student stipend support for HS received from Merck/AAAS; support for KH received from the Arthur Vining Davis Foundation.