Neurons and muscles recorded
This protocol is extracted from research article:
A central control circuit for encoding perceived food value
Sci Adv, Nov 21, 2018; DOI: 10.1126/sciadv.aau9180

The AJM is a large thick muscle that is involved in the retraction of the buccal mass and the radula/odontophore complex (18). Electromyography (EMG) recordings were obtained from the anterior region of this muscle. Most of the activity recorded occurred during the retraction phase (18). The SLRT muscles are the largest of the tensor muscles and the bulkiest in the odontophore and have been previously reported to be involved in the retraction phase of a cycle (18). EMG recordings were performed on the SLRT on the dorsolateral edges of the odontophore. Command-like interneuron CV1a is located in the cerebral ganglia and was identified by its electrical properties, characteristic location, and ability to drive fictive feeding cycles when artificially depolarized to fire spikes. The N2v neuron is a CPG interneuron located on the ventral surface of the buccal ganglia. It can be identified by its characteristic plateau during the retraction phase of a cycle. Artificial activation of an N2v caused widespread retraction phase activity in many buccal neurons and activity on the AJM. B11 is a newly identified SLRT motoneuron located on the ventral surface of the buccal ganglia. B11 was identified using morphological and electrophysiological criteria. The LBNs and VBNs contain the axons of motoneurons innervating the SLRT. We therefore backfilled these nerves with 5(6)-carboxyfluorescein (5-CF). This reveals a population of putative SLRT motoneurons that we could reidentify in other preparations and test electrophysiologically. Using the preparation consisting of the CNS connected to the SLRT muscle via the lateral and ventral buccal nerves, neurons of interest were impaled, stimulating them while recording extracellular potentials in the SLRT. Spikes in B11 caused contraction of the SLRT muscle and robust 1:1 responses were recorded on the muscle. Touch to the esophagus initiated a barrage of EPSPs on B11, which caused it to spike in the protraction phase of the initiated cycle. To identify candidate members of the egestion network, neurons had to fulfill two criteria: be active in sensory-driven egestion cycles and be sufficient to drive egestion cycles. Projection interneurons are known to be influential in driving patterned activity in Lymnaea (15). Backfilling the CBC with 5-CF revealed a population of projection neurons, including the paired PRNs. PRN can both drive fictive egestion cycles and is activated during cycles initiated by touch to the esophagus, fulfilling both criteria. PRN also has a 1:1 excitatory connection with B11. RM is a newly identified mechanosensory neuron in the buccal ganglia. Touch to the radula causes somatic spikes in RM, which rise from the baseline and persist in a saline containing zero Ca2+ and EGTA, blocking chemical synaptic transmission. The saline contained 35.0 mM NaCl, 1.6 mM KCl, 18.0 mM MgCl2, 2.0 mM EGTA, and 10 mM Hepes buffer in water. vTN was identified due to its location and white color and response to tactile stimulation of the radula (17).

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