An identified glutamatergic interneuron patterns feeding motor activity via both excitation and inhibition.

Abstract

1. Previously we demonstrated that glutamate is an important neurotransmitter in the CNS of Helisoma. Exogenous glutamate applied to the buccal ganglia mimicked both the excitatory and inhibitory effects of subunit 2 (S2) of the tripartite central pattern generator (CPG) on S2 postsynaptic motor neurons. Here we identify buccal interneuron B2 as an S2 interneuron by utilizing a combination of electrophysiology, pharmacology, and intracellular staining. In addition, neurons that were electrophysiologically and morphologically characterized as neuron B2 demonstrated antiglutamate immunoreactivity, suggesting that neuron B2 is a source of endogenous glutamate in the buccal ganglia. 2. Depolarization of neuron B2 evoked excitatory postsynaptic potentials in motor neurons excited by S2. The excitatory effects of B2 depolarization and S2 activation were reversibly antagonized by the ionotropic glutamate receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione, similar to the antagonism shown previously for application of exogenous glutamate. Depolarization of neuron B2 also evoked inhibitory postsynaptic potentials in motor neurons inhibited by S2. When such motor neurons were maintained in isolated cell culture, application of exogenous glutamate produced a direct hyperpolarization of the membrane potential. 3. The activity of neuron B2 is necessary for the production of the standard pattern of buccal motor neuron activity, which underlies functional feeding movements. The subunits of the tripartite buccal CPG must be active in the temporal sequence S1-S2-S3 to produce the standard feeding pattern. Rhythmic inhibition from neuron B2 terminated activity in S1 postsynaptic motor neurons and entrained the frequency of activity in S3 postsynaptic motor neurons. Hyperpolarization of neuron B2 disrupted the production of the standard motor pattern by eliminating S2 postsynaptic potentials in identified buccal motor neurons, thereby prolonging S1 activity and disrupting S3 bursting. 4. These data support the hypothesis that S2 neuron B2 is glutamatergic and demonstrate that glutamatergic transmission, and especially inhibition, is fundamental to the production of behaviorally critical motor neuron activity patterns in Helisoma.