Studies using the third instar larval neuromuscular junction (NMJ) to examine the effects of altering CaMKII on neuronal activity found major effects on motor neuron excitability; inhibition of CaMKII caused hyperexcitability (Wang et al., 1994, Griffith et al., 1994), while constitutive CaMKII caused failures and delayed action potentials (Park et al., 2002). The nature of the NMJ preparation (no neurons, just terminals), however, made investigation of the role of CaMKII in the plasticity of intrinsic properties difficult. In the last several years we have developed new electrophysiological preparations utilizing whole-cell patch clamp of neurons identified by their cell-specific expression of GFP. These technical advances make it possible, for the first time in the fly, to study identified central neurons in the intact larval and adult brains (Choi et al., 2003; Park and Griffith, 2006).

Recording from Identified Third Instar Larval Motor Neurons

We have used dye-fills and electrophysiological recordings to identify and characterize a cluster of motor neurons in the third instar larval ventral ganglion. This cluster of neurons is similar in position to the well-studied embryonic RP neurons. The terminal targets of these five neurons are body wall muscles 6/7, 1, 14, 30 and the intersegmental nerve (ISN) terminal muscles (1, 2, 3, 4, 9, 10, 19, 20). All cells except the ISN neuron, which has a type Is ending, display type Ib boutons. Current clamp recordings shoe that the MNISN-Is neuron has a longer delay in the appearance of the first spike compared to type Ib neurons. Genetic, biophysical and pharmacological studies in current and voltage clamp show this delay is controlled by the kinetics and voltage-sensitivity of inactivation of the Shal I_A current. Tetanic stimulation of the MNISN-Is neuron can elicit long-term changes in the delay to first spike and the firing rate. We are currently investigating the molecular basis of these changes in intrinsic properties. The combination of genetic identification and whole cell recording allows us to directly explore the cellular substrates of neural and locomotor behavior in an intact system.

Recording from Adult Circadian Pacemaker Cells

To study the properties and modulation of neurons involved in an adult behavior, we have recorded from the large ventrolateral neurons (LN_vs) of the adult brain. These neurons have been suggested to be the core of the circadian clock and are readily identified with a neuron subtype-specific GAL4 line (pdf-GAL4) driving mCD8-GFP. Work from many groups has suggested that regulation of the excitability of these neurons is important to their circadian function. Fills of the 4 large LNvs indicate that they are anatomically equivalent with projections to both the contra- and ipsilateral medullas. Current clamp recordings show a stereotyped response of this cell type to current injection with action potentials evoked with minimal current injection (4-10 pA). Resting membrane potential upon breaking into the cell varied between -40 mV and -65 mV, and appeared to be light-sensitive at the beginning of the day. These studies demonstrate that the cellular basis of adult behavior can be approached electrophysiologically in the Drosophila CNS.

 

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