Ishani Das

Mentor: Peter Brodfuehrer

The medicinal leech, Hirudo verbana, has long been used as a model system for investigation the neuronal basis of rhythmic behavior, such as swimming. Leech swimming is an episodic behavior which can be elicited by brief (1 s) stimulation of the body wall, producing a single swim bout lasting 10-30 seconds. Prior research has elucidated a neuronal network, at the level of interactions between individual neurons, how the nervous system initiates and generates leech swimming behavior. However, the neuronal mechanism underlying the transition from brief stimulation to sustained excitation in several neurons involved in generating swimming is still unknown. Previous research has shown that one class of glutamate receptors (non-NMDA) are critical for sustaining excitation in the neuronal network controlling leech swimming.

In many other systems, sustained excitation in neurons occurs via calcium influx through glutamate receptors. Glutamate receptors are composed of four subunit subtypes, GluA1-4, which occur primarily as heterotetramers (two identical pairs of subunits), and are calcium impermeable when GluA2 is present. GluA2 makes the receptor calcium impermeable due to post-translation RNA editing in which a glutamine (Q) codon in the pore-lining region is substituted for an arginine (R) codon.

Using molecular techniques, the Brodfuehrer laboratory has cloned one non-NMDA receptor subunit on (nNL2) and three partial subunits (nNL1, 3 and 4). Protein sequences of these subunits show differences in Q/R editing at the hypothesized pore region, suggesting that some glutamate receptors subunits in leech neurons may be calcium permeable. Two approaches are being used to determine whether non-NMDA receptors in the leech nervous system are calcium permeable.  First, we plan to use RACE (rapid amplification of cDNA ends) protocol to obtain complete coding sequences of nNL1, 3, and 4.  Once a complete clone of at least one other subunit is obtained, we will use the Xenopus oocyte expression system to characterize nonNMDA receptor’s calcium permeability. Second, we will determine if neurons comprising the swim network contain calcium permeable nonNMDA channels using a fluorescently-labeled antagonist to calcium permeable nonNMDA.   Neurons in the swim network will first be labeled by injecting a fluorescent dye into the cell.  The nervous system will then be bathed in the fluorescently-labeled antagonist and any overlap between the two fluorescent labels will indicate that calcium permeable nonNMDA receptors are located on that particular neuron in the swim network.  Establishing the physiological characteristics and localization of the nonNMDA receptors will add to our understanding of the important role these receptors play in the transition from brief stimulation to sustained excitation in the leech swim network.