Using voltage sensitive dyes to study connectivity among neurons during development of sensory-motor circuits in the leech.

ANTONIA MARIN BURGIN; W. B. KRISTAN JR; K.A. FRENCH

San Diego

Congreso; Meeting of the society for neuroscience; 2004

Society for Neuroscience

The neural circuit that produces local bending behavior in the leech Hirudo medicinalis consists of 3 layers of neurons: 4 mechanosensory P cells, at least 17 identified interneurons, and about 24 inhibitory and excitatory motor neurons that innervate longitudinal muscles. All connections between layers are excitatory. Inhibition resides in the motor neuron layer, allowing the animal to contract on one side while relaxing the opposite side. Local bending behavior arises at about 60%  of embryonic development (60% ED). To study the development of connections within and between the layers, we applied FRET voltage-sensitive dyes onto isolated ganglia from leech embryos of different developmental stages and from adults. We impaled one neuron while imaging approximately 50 neurons. Images were recorded while an impaled P cell or motor neuron (MN) was stimulated. Coherence analysis detected neurons with a time-locked response to the stimulated cell. In ganglia from embryos at 58% ED, the cells driven by the stimulated P cell or MN were in-phase with the driven neuron, demonstrating the extent of the electrical connections between neurons at that stage. In ganglia from embryos at 70% ED or from adults, responses in cells driven by the stimulated P cell showed a variety of phase locking to the driven neuron, revealing inhibitory, as well as excitatory synapses. When an inhibitory MN (iMN) was driven, one group of neurons-the electrically coupled ones-were in-phase with the driven cell, and another group of cells were out-of-phase with it, indicating that they were inhibited by this iMN. These results indicate that electrical connections are present in this circuit from before the appearance of local bending into adulthood, but the mature circuit acquires inhibition in the motor layer just at the time when local bending behavior appears.