Decoding the intensity of sensory input by two glutamate receptors in one C. elegans interneuron

How neurons are capable of decoding stimulus intensity and translate this information into complex behavioral outputs is poorly defined. Here, we demonstrate that the C. elegans interneuron AIB regulates two types of behaviors: reversal initiation and feeding suppression in response to different concentrations of quinine. Low concentrations of quinine are decoded in AIB by a low-threshold, fast-inactivation glutamate receptor GLR-1 and translated into reversal initiation. In contrast, high concentrations of quinine are decoded by a high-threshold, slow-inactivation glutamate receptor GLR-5 in AIB. After activation, GLR-5 evokes sustained Ca2+ release from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores and triggers neuropeptide secretion, which in turn activates the downstream neuron RIM and inhibits feeding. Our results reveal that distinct signal patterns in a single interneuron AIB can encode differential behavioral outputs depending on the stimulus intensity, thus highlighting the importance of functional mapping of information propagation at the single-neuron level during connectome construction.


Nematode strains
The following worm strains were used: WT: N2 (the wild type).

Molecular Biology
Promoters labeling specific neurons are described as below: AIB: npr-9, RIM: gcy-13 or cex-1, ASH: sra-6. The promoters were PCR-amplified from N2 genomic DNA. These PCR fragments were recombined with the Hind III and BamH I, Sph I and BamH I, Xba I and BamH I sites in the modified donor vectors, respectively. In this way, AIB::GCaMP3.0, AIB::ChR2, RIM::GCaMP1.3, and ASH::GCaMP3.0 plasmids were generated. Neuronspecific RNAi was generated as previously described 1  The expression pattern of glr-5 was conducted by a fosmid from Source BioScience with Clone Transgene Ome Resource: CBGtg9050H043D, which contain the full length segment of glr-5 and about 15 kb upstream and 10 kb downstream sequence, attached with a GFP sequence before its stop codon. This fosmid was co-injected with a plasmid Pnpr-9::mkate2, which is specifically expressed in AIB neuron.

Behavioral Assays for Reversal
To calculate reversal index, a previously described protocol of the 'drop test' assay is employed 2 . In brief, a drop of quinine solution was delivered near the tail of a forwardmoving animal. When the repellent quinine was concentrated enough, the animal ceases forward movement and reverses; if quinine was diluted sufficiently, extremely just water alone, the animal will continue moving forward. The response of a single animal to each drop delivered was recorded as either a positive or a negative response. Response was considered positive when the avoidance reflex was observed within 4 s after the animal encountered quinine. The results of the reversal assays were represented as a reversal index, which was defined as the number of positive responses divided by the total number of trials (drops delivered). At least 10 worms a group were tested to calculate a reversal index, and more than 6 groups were tested per experiment. Every experiment was repeated at least three times. The assay was conducted always on unseeded NGM plates. An interval of 10 min was used between successive drops challenged to the same animal.
Each animal was tested with no more than 5 successive drops per day. A schematic drawing of the iCaN system, which was developed to simultaneously analyze the locomotion and feeding behaviors while monitoring intracellular Ca 2+ concentration ([Ca 2+ ]i) at single neuron resolution on freely moving worms. A low-magnification objective on top of the sample was used to track the movement of free-moving worms and to record the feeding behavior. A high-magnification objective from the bottom to monitor the neuronal [Ca 2+ ]i activities at the single neuron resolution. The recorded locomotion is feedback to x-y stage to center the worm in the field-of-view of both objectives. Special z stacking design is undertaken to ensure fast 4D imaging of worm neurons at high resolution. Changes in fluorescence of the calcium indictor GCaMP were monitored with an EMCCD camera. Fluorescence of mKate2 was acquired as reference in most experiments.

Supplementary Figures and Figure Legends
To simultaneously monitoring GCaMP fluorescence and turning on ChR2 or NpHR, a LED light source emitting blue and yellow light was added to the iCaN system. Pharyngeal