The Input-Output Relationship of AIY Interneurons in Caenorhabditis elegans in Noisy Environment

Abstract

Determining how neurotransmitter input causes various neuronal activities is crucial to understanding neuronal information processing. In Caenorhabditis elegans, AIY interneurons receive several sources of sensory information as glutamate inputs and regulate behavior by integrating these inputs. However, the relationship between glutamate input and the Ca²⁺ response in AIY under environmental noise, in other words, without explicit stimulation, remains unknown. Here, we show that glutamate-input fluctuations evoke a sporadic Ca²⁺ response in AIY without stimulation. To ensure that Ca²⁺ response can be considered AIY output, we show that the membrane-potential depolarization precedes Ca²⁺ responses in AIY. We used an odor as model stimulation to modulate the sensory inputs. Simultaneous imaging of glutamate input and Ca²⁺ response, together with glutamate transmission mutants, showed that glutamate-input fluctuations evoke sporadic Ca²⁺ responses. We identified the input-output relationships under environmental noise in vivo, and our results address the relationship between sensory-input fluctuations and behavioral variability.

Keywords: Neuroscience; Sensory Neuroscience; Systems Neuroscience.

Graphical abstract

Figure 1

Simultaneous Imaging of Membrane Potential and Ca²⁺ in AIY Interneurons (A) Confocal image of the voltage indicator (ArcLight: green, left) and Ca²⁺ indicator (R-GECO magenta, middle). The merged image is on the right. Scale bar, 10 μm. A, anterior; L, left; P, posterior; R, right. (B) Representative results of Ca²⁺ (red) and membrane potentials (black) at the soma (left) and neurite (right). (C) Averaged Ca²⁺ spike (red) and membrane-potential spike (black) without (left) and with (right) odor. The shadows around the solid lines indicate the standard error of the mean (SEM) (no odor, N = 20, n = 16; odor, N = 10, n = 15). (D) Membrane potential (left) and Ca²⁺ spike (right) intensities, with (filled boxes) and without (hollow boxes) odor. Box plots include the median (center line), quartiles (boxes), and range (whiskers). The statistical metrics are as follows: membrane potential, p = 0.38; Ca²⁺, p = 0.85; paired t tests (no odor, N = 20, n = 16; odor, N = 10, n = 15). (E) Correlation between Ca²⁺ spike amplitude and membrane-potential spike amplitude. The red diamonds and pink dots are the responses in the presence and absence of odor stimulation, respectively. The statistical metrics are as follows: no odor, Pearson correlation coefficient (PCC) = −0.19, p = 0.48; odor, PCC = −0.40, p = 0.14 (no odor, N = 20, n = 16; odor, N = 10, n = 15).

Figure 2

Simultaneous Imaging of Glutamate Inputs and Ca²⁺ Responses in AIY Interneurons (A) Confocal image of the glutamate indicator (iGluSnFR: green, left) and Ca²⁺ indicator (R-GECO: magenta, middle) in AIY. The merged image is on the right. Scale bar, 10 μm. A, anterior; L, left; P, posterior; R, right. (B) Averaged glutamate input (blue) and Ca²⁺ response (red) to odor. The shaded region indicates isoamyl alcohol (IAA) stimulation, and the shadows around the solid lines indicate the SEM. (C) Averaged glutamate intensities before (pre-addition) and after (post-addition) odor addition (left). Glutamate intensities before (pre-removal) and after (post-removal) odor stimulation removal (right). The statistical metrics are as follows: left, p = 0.00010; right, p = 0.000086; paired t test (N = 15). ***p < 0.001.

Figure 3

Glutamate Input When the Ca²⁺ Spike Occurs (A) Representative responses of Ca²⁺ and glutamate input (left). Magnifications of the dotted boxes showing the responses without (middle) and with (right) odor stimulation. The blue and red lines are the glutamate input and Ca²⁺ response, respectively. The shaded region indicates IAA stimulation. (B) Definition of the response categories. (C) The averaged glutamate responses coincident with Ca²⁺ spikes. Time 0 indicates the onset of Ca²⁺ spikes. All glutamate responses were normalized to zero at time 0. The purple, blue, and green lines indicate the responses under the no odor, early odor, and later odor conditions, respectively (defined in B). The error bars indicate the SEM (N = 15; no odor, n = 15; early odor, n = 11; later odor, n = 21). (D) Glutamate inputs before the Ca²⁺ spike and at spike onset. “Pre” indicates the average intensity from −3 to −2 s in (C), and “onset” indicates the average intensity from −0.5 to +0.5 s. The statistical metrics are as follows: no odor, p = 0.010; early odor, p = 0.0024; later odor, p = 0.0037; paired t test (N = 15; no odor, n = 15; early odor, n = 11; later odor, n = 21). *p < 0.05, **p < 0.01. (E) Correlation between glutamate decreases and Ca²⁺ increases. The purple dots, blue diamonds, and green triangles are correlations under the no odor, early odor, and later odor conditions, respectively. The statistical metrics are as follows: no odor, PCC = 0.58, p = 0.022; early odor, PCC = 0.080, p = 0.81; later odor, PCC = 0.069, p = 0.77 (no odor, n = 15; early odor, n = 11; later odor, n = 21). (F) Glutamate decreases with and without Ca²⁺ spikes. The filled and hollow box plots indicate decreases with and without a spike, respectively. Box plots indicate the median (center line), quartiles (boxes), and range (whiskers). The statistical metrics are as follows: no odor, p = 0.0062; later odor, p = 0.037; Welch's t test (N = 15; no odor, spike, n = 15; no odor, no spike: n = 89; later odor, spike, n = 21; later odor, no spike, n = 69) *p < 0.05, **p < 0.01.

Figure 4

Ca²⁺ Response to Decreases in Glutamate Input (A) Ca²⁺ response to decreases in glutamate input. Time 0 indicates the time when the glutamate decreases. All Ca²⁺ responses were normalized to zero at time 0. The red and yellow lines indicate the response under the no odor and later odor conditions, respectively. The shadows indicate the SEM (for the Ca²⁺ response under later odor condition, the shadows are too small to see; N = 15; no odor, n = 43; later odor, n = 61). (B) Ca²⁺ intensities before (“pre”; averaged from −3 to −2 s in A) and after (“onset”; averaged from −0.5 to +0.5 s in A) glutamate decrease. The statistical metrics are as follows: no odor, p = 0.014; later odor, p = 0.014; paired t test (N = 15; no odor, n = 43; later odor, n = 61). *p < 0.05. (C) Ca²⁺ responses to decreases in glutamate input relative to randomly selected data with (right) and without odor (left). The gray lines indicate the averaged randomly selected data ± 3 SD (see Methods). (D) Ca²⁺ increases with glutamate decreases (colored boxes) and by random selection (gray boxes). Box plots indicate the median (center line), quartiles (boxes), and range (whiskers). The statistical metrics are as follows: no odor, p = 0.045; later odor, p = 0.0015. Wilcoxon rank-sum test (N = 15; no odor, decrease, n = 43; no odor, random, n = 100; later odor, decrease, n = 61; later odor, random, n = 100). *p < 0.05, **p < 0.01.

Figure 5

Ca²⁺ Responses and Glutamate Inputs in glc-3 Mutants (A) Averaged glutamate inputs (blue) and Ca²⁺ responses (red) to odor. The shaded region indicates IAA stimulation, and the shadows around the solid lines indicate the SEM (N = 12). (B) Glutamate intensities before (pre-addition) and after (post-addition) odor stimulation onset (left), and average intensities before (pre-removal) and after (post-removal) odor stimulation removal (right). The statistical metrics are as follows: left, p = 0.0030; right, p = 0.0018; paired t test (N = 12). **p < 0.01. (C) Averaged glutamate responses coincident with Ca²⁺ spikes. Time 0 indicates the onset of Ca²⁺ spikes. All glutamate responses were normalized to zero at time 0. The purple, blue, and green lines indicate the responses under the no odor, early odor, and later odor conditions, respectively. Shadows indicate the SEM (N = 12, no odor, n = 15; early odor, n = 6; later odor, n = 20). (D) Glutamate input before the Ca²⁺ spike and at spike onset. “Pre” indicates the average intensity from −3 to −2 s in (C), and “onset” indicates the average intensity from −0.5 to +0.5 s. The statistical metrics are as follows: no odor, p = 0.0012; early odor, p = 0.65; later odor, p = 0.89; paired t test (N = 12, no odor, n = 15; early odor, n = 6; later odor, n = 20). **p < 0.01. (E) Averaged glutamate decreases with (filled boxes) and without (hollow boxes) Ca²⁺ spikes. Box plots indicate the median (center line), quartiles (boxes), and range (whiskers). The statistical metrics are as follows: no odor, p = 0.0027; later odor, p = 0.58; Welch's t test (N = 12, no odor, spike, n = 15; no odor, no spike, n = 114; later odor, spike, n = 20; later odor, no spike, n = 82). **p < 0.01.

Figure 6

Ca²⁺ Responses and Glutamate Inputs in eat-4 Mutants (A) Averaged glutamate input (blue) and Ca²⁺ response (red) to odor. The shaded region indicates IAA stimulation and the shadows around the solid lines indicate the SEM (N = 12). (B) Average glutamate intensity before (pre-addition) and after (post-addition) odor stimulation onset (left), and average intensity before (pre-removal) and after (post-removal) odor stimulation removal (right). Error bars indicate the SEM. The statistical metrics are as follows: left, p = 0.41; right, p = 0.055; paired t test (N = 12). (C) Ca²⁺ spike probability following odor addition. Comparison between N2 animals and glc-3 mutants: p = 0.13; comparison between N2 animals and eat-4 mutants: p = 0.014. Fisher's exact test with Holm correction (N2, N = 15; glc-3, N = 12; eat-4, N = 12); *p < 0.05. (D) Ca²⁺ spike frequency in N2 (filled), glc-3 (striped), and eat-4 mutants (hollow) with and without odor. Box plots indicate the median (center line), quartiles (boxes), and range (whiskers). The statistical metrics are as follows: N2 data comparison between no odor and later odor, p = 0.0045; glc-3 data comparison between no odor and later odor, p = 0.34; eat-4 data comparison between no odor and later odor, p = 0.34; Wilcoxon signed rank test. No odor data comparison between N2 and glc-3, p = 0.024; no odor data comparison between N2 and eat-4, p = 0.00044; later odor data comparison between N2 and glc-3, p = 0.014; later odor data comparison between N2 and eat-4, p = 0.000015; Wilcoxon rank-sum test. The Holm method was used for the correction (N2, N = 15; glc-3, N = 12; eat-4, N = 12). *p < 0.05, **p < 0.01, ***p < 0.001. (E) Averaged glutamate responses coincident with Ca²⁺ spikes. Time 0 indicates the onset of the Ca²⁺ spike. All glutamate responses were normalized to zero at time 0. The purple, blue, and green lines indicate the responses under the no odor, early odor, and later odor conditions, respectively. The bars indicate the SEM (N = 12, no odor: n = 9; early odor, n = 3; later odor, n = 9). (F) Glutamate input before Ca²⁺ spikes and at Ca²⁺ spike onset. “Pre” indicates the average intensity from −3 to −2 s in (E), and “onset” indicates the average intensity from −0.5 to +0.5 s. The statistical metrics are as follows: no odor: p = 0.60; early odor, p = 0.53; later odor, p = 0.50; paired t test (N = 12; no odor, n = 9; early odor, n = 3; later odor, n = 9). (G) Averaged glutamate decreases (filled boxes) with and without (hollow boxes) Ca²⁺ spikes. Box plots indicate the median (center line), quartiles (boxes), and range (whiskers). The statistical metrics are as follows: no odor, p = 0.88; later odor, p = 0.48; Welch's t test (N = 12, no odor, spike, n = 9; no odor, no spike, n = 108; later odor, spike, n = 9; later odor, no spike, n = 100).