1 and mCD8-GFP in a subset of E564 neurons learn more (

Gordon and Scott, 2009). Adult flies were then assayed for constitutive extension, and the frequency distributions of cell-types in extenders and nonextenders were compared ( Figure 2C). Cell-type 1 was highly enriched in extenders and rarely labeled in nonextenders, whereas the other cells were present at similar frequencies in extenders and nonextenders. Additionally, in five animals that displayed constitutive proboscis extension, cell-type 1 was exclusively labeled, demonstrating that silencing of these neurons produces the aberrant behavior. The neurons that inhibit proboscis extension (which we name PERin) have cell bodies and processes in the first leg neuromeres of the VNC and projections to the SOG, the brain region that contains gustatory sensory axons and proboscis motor neuron dendrites (Figures 2D–2G). Labeling with the presynaptic synaptotagmin-GFP marker (Zhang et al., 2002) and the postsynaptic DenMark marker (Nicolaï et al., 2010) indicated that the dendrites of PERin neurons are restricted to the first leg neuromeres, whereas axons are

found in both the SOG and the first leg neuromeres (Figures 2H and 2I). The anatomy of these neurons suggests that they convey information from the leg neuromeres to a region of the fly brain involved in gustatory processing and proboscis extension. Anatomical studies examining the proximity of PERin fibers to gustatory sensory dendrites or proboscis motor axons revealed that PERin neurons do not come into check details close contact with known neurons that regulate proboscis extension (Figure S2; Movies S1 and S2). There are several different contexts in which PERin neurons might modulate feeding initiation. PERin activity might reflect the satiety the state of the animal, such that high activity inhibits feeding initiation when the animal is fed and low activity promotes feeding when the animal is food-deprived. Alternatively, PERin

neurons might directly process gustatory sensory cues, increasing activity in response to bitter compounds to suppress proboscis extension or decreasing activity upon sucrose stimulation to promote extension. A third possibility is that they regulate proboscis extension in response to other cues, such as mechanosensory or somatosensory cues, to inhibit proboscis extension while the animal is engaged in other behaviors. To test whether PERin neurons influence extension probability based on satiety state, we performed cell-attached electrophysiological recordings to monitor the basal firing rate of PERin neurons under fed and food-deprived conditions (Marella et al., 2012). In both conditions, PERin neurons exhibited constant basal activity of ∼14 Hz, indicating that tonic activity in these neurons is not altered by satiety state (Figures 3A and 3B).