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[Preprint]. 2023 Sep 25:2023.09.11.557249.
doi: 10.1101/2023.09.11.557249.

Programmed Cell Death Modifies Neural Circuits and Tunes Intrinsic Behavior

Alison Kochersberger  1 , Mohammad Mahdi Torkashvand  2 , Dongyeop Lee  3 , Saba Baskoylu  4 , Titas Sengupta  5 , Noelle Koonce  5 , Chloe E Emerson  1 , Nandan V Patel  1 , Daniel Colón-Ramos  5   6   7 , Steven Flavell  4 , H Robert Horvitz  3 , Vivek Venkatachalam  2 , Marc Hammarlund  1
Affiliations

Programmed Cell Death Modifies Neural Circuits and Tunes Intrinsic Behavior

Alison Kochersberger et al. bioRxiv. .

Abstract

Programmed cell death is a common feature of animal development. During development of the C. elegans hermaphrodite, programmed cell death (PCD) removes 131 cells from stereotyped positions in the cell lineage, mostly in neuronal lineages. Blocking cell death results in supernumerary "undead" neurons. We find that undead neurons can be wired into circuits, can display activity, and can modify specific behaviors. The two undead RIM-like neurons participate in the RIM-containing circuit that computes movement. The addition of these two extra neurons results in animals that initiate fewer reversals and lengthens the duration of those reversals that do occur. We describe additional behavioral alterations of cell-death mutants, including in turning angle and pharyngeal pumping. These findings reveal that, like too much PCD, too little PCD can modify nervous system function and animal behavior.

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Conflict of interest statement

Competing interests Authors declare that they have no competing interests.

Figures

Fig. 1:
Fig. 1:. Undead RIM sister cell form synapses with AIB, altering presynaptic patterning.
(A-G) Fate marker analysis in the ced-3(n717) (cell-death mutant) background of selected neuron types with undead relatives. Each panel shows the percentage of animals exhibiting fewer or more than the ‘expected number’ of cells, as well as a representation of the local cell lineage (1, 2). The ‘expected number’ is defined as the number of neurons of the type present in wild-type animals, plus the number of lineage-proximate undead cells. (H) Micrograph of RIM fate marker in wild-type and ced-3(n717) (cell-death mutant) backgrounds. Arrowheads indicate cell bodies. The wild-type animal has two RIM neurons, and the ced-3(n717) animal has two additional RIM-like cells and is counted as contributing to the orange “more than expected” sector in panel (A). 10μm scalebars. (I) Schematic of AIB synaptic connections to RIM and other neurons (24). (J) Quantification of animals with stereotyped AIB synaptic patterning in wild-type and ced-3(n717) animals. (K) RAB-3::GFP pre-synapses in AIB (top panel) and GLR-1::tagRFP post-synapses in RIM (middle panel) colocalize (bottom panel). (L) ced-3(n717) mutants display an increase in AIB pre-synapses (top panel) and RIM post-synapses (middle panel) when both RIM and RIM-like cells are labeled. (M) ced-3(n717) mutants display an increase in AIB pre-synapses (top panel) but display normal RIM post-synapses (middle panel) when only one of the RIM and RIM-like cells is labeled because of mosaicism of the labeling array. 2μm scalebars.
Fig. 2.
Fig. 2.. Undead RIMs are functional and their activity corresponds to reversal behavior.
(A) Annotated micrographs of RIM neurons in wild-type and ced-3(n717) animals labeled with GCaMP6s and tagRFP. Red boxes show the cell-body regions that were analyzed. Four cells are boxed for ced-3(n717) animals: the two RIM neurons (RIML and RIMR) and the two undead RIM-like cells (RIMLU and RIMRU). (B) Calcium traces of RIM neurons in a wild-type animal. Dashed vertical lines represent reversal events. (C) Calcium traces of RIM and RIM-like neurons in a ced-3(n717) animal. Dashed vertical lines mark reversal events. (D) Heatmap of calcium traces of RIM and RIM-like neurons across all ced-3(n717) animals. Each row corresponds to a RIM (L, R) or RIM-like (LU, RU) neuron, with its name on the left side of the row. Individual worms are grouped within horizontal black lines. Dashed vertical lines mark reversal events. (E) Averaged calcium traces in specific neurons in ced-3(n717) animals, aligned on reversal events (dashed vertical lines). (F) Correlation coefficients between different pairs of specific neurons in ced-3(n717) animals.
Fig. 3.
Fig. 3.. Undead RIMs alter reversal frequency and duration.
(A) ced-3(n717) and ced-4(n1162) animals initiate fewer reversals than wild-type animals. Data are mean ± S.D. with individual data points shown. (wild-type n=41, ced-3(n717) n=42, ced-4(n1162) n=42) (B) Reversal durationis increased in ced-3(n717) and ced-4(n1162) animals. Reversal times measured from events in Fig 3A. (C) Schematic of behavioral experiment with RIM-specific cell death rescue. (D) ced-3(n717) animals initiate fewer reversals than wild-type animals, and removal of additional RIMs partially rescues reversals. Data are mean ± S.D. with individual data points shown. Wild-type, n=58; ced-3(n717), n=58; Pinx-19::ced-3A (4RIM), n=49; Pinx-19::ced-3A (2RIM), n=29. (E) Reversal length is increased in ced-3(n717) animals, and this increase is partially rescued by RIM-specific re-introduction of functional CED-3A using the inx-19 promoter. Data are mean ± S.D. with individual data points shown. Reversal times measured from events in Fig 3D.
Fig. 4.
Fig. 4.. Analysis of cell death mutant behavior.
(A-B) Worm tracks on bacterial lawn. A wild-type worm moves straight (A), while a ced-3(n717) knockout worm tends to turn (B). (C-D) Quantification of the turning angle between control and ced-3(n717) (C) and between control and ced-4(n1162) (D). Worm strains used in panel D contain nIs177[Pceh-28::4xNLS::GFP]. Data are mean ± S.D with N=20.
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