Presynaptic coupling by electrical synapses coordinates a rhythmic behavior by synchronizing the activities of a neuron pair

Abstract

Electrical synapses are specialized structures that mediate the flow of electrical currents between neurons and have well known roles in synchronizing the activities of neuronal populations, both by mediating the current transfer from more active to less active neurons and by shunting currents from active neurons to their less active neighbors. However, how these positive and negative functions of electrical synapses are coordinated to shape rhythmic synaptic outputs and behavior is not well understood. Here, using a combination of genetics, behavioral analysis, and live calcium imaging in Caenorhabditis elegans, we show that electrical synapses formed by the gap junction protein INX-1/innexin couple the presynaptic terminals of a pair of motor neurons (AVL and DVB) to synchronize their activation in response to a pacemaker signal. Live calcium imaging reveals that inx-1/innexin mutations lead to asynchronous activation of AVL and DVB, due, in part, to loss of AVL-mediated activation of DVB by the pacemaker. In addition, loss of inx-1 leads to the ectopic activation of DVB at inappropriate times during the cycle through the activation of the L-type voltage-gated calcium channel EGL-19. We propose that electrical synapses between AVL and DVB presynaptic terminals function to ensure the precise and robust execution of a specific step in a rhythmic behavior by both synchronizing the activities of presynaptic terminals in response to pacemaker signaling and by inhibiting their activation in between cycles when pacemaker signaling is low.

Keywords: C. elegans; electrical synapse; innexin; lateral excitation; shunting inhibition.

Fig. 1.

inx-1 functions in AVL and DVB motor neurons to regulate the frequency and timing of expulsion during the DMP. (A) Model for the circuit regulating expulsion (Exp). Calcium oscillations in the intestine (pacemaker) every 50 s lead to SNT-2/synaptotagmin-dependent secretion of NLP-40 from dense core vesicles (DCVs). NLP-40 activates the GPCR, AEX-2, in AVL and DVB, leading to calcium spike generation through VGCCs and GABA release from NMJs, which leads to the enteric muscle contraction. AVL and DVB NMJs are functionally coupled by INX-1/innexin, which coordinates AVL/DVB activities by suppressing ectopic calcium influx and promoting NLP-40-dependent AVL/DVB activation. (B) Quantification of the number of Exp per DMP cycle in adult worms with the indicated genotypes. “Exp per cycle” denotes the ratio of Exp per pBoc, which defines the start of the DMP. “Normal” denotes Exp occurring less than 5 s after pBoc, and “Ectopic” denotes Exp occurring more than 5 s after pBoc. DVB miniSOG and AVL miniSOG denote transgenes expressing miniSOG under control of a flp-10 and flp-22 promoter fragment, respectively. (C) Histograms showing the time when each Exp occurred after pBoc in the indicated strains. The average Exp time after pBoc with SEs is shown for wild-type, DVB miniSOG, AVL miniSOG, and inx-1 mutants. (D) Quantification of the number of Exp per DMP cycle in adult nlp-40 mutants, nlp-40; inx-1 mutants or nlp-40; inx-1 mutants expressing the indicated transgenes. “intestinal inx-1” denotes full length inx-1a cDNA expressed under the intestine-specific nlp-40 promoter. “neuronal inx-1” denotes inx-1a cDNA expressed in GABAergic neurons using the unc-47 promoter. “muscle inx-1” denotes inx-1a cDNA expressed in body wall muscles using the myo-3 promoter. “heat shock inx-1” denotes inx-1a cDNA expressed using the heat shock promoter (Phsp-16.2) either without or with heat shock for 1 h at 34 °C. DVB inx-1 and AVL inx-1 denote transgenes expressing inx-1a cDNA under control of the flp-10 and flp-22 promoter, respectively. (E) (Above) Diagram showing the cell bodies and axons of AVL/DVB and the NMJ region of AVL/DVB in the preanal ganglia. (Below) Transmission electron micrograph image of AVL and DVB axons in cross section in the preanal ganglion region (image JSE_207, also known as JSE_122116), showing a large gap junction (arrowhead) between AVL and DVB axons. The gap junction appears as electron dense areas within the plasma membranes of AVL and DVB where they contact each other. DD6 refers to the soma of the DD6 motor neuron. The electron dense regions of AVL and DVB plasma membranes are present in eight serial sections (JSE_205 to JSE_213). Adapted with permission from David Hall, Albert Einstein College of Medicine. Means and SEs are shown. Student’s t test: ^***P < 0.001; ^##P < 0.001 compared to wild type; ^#P < 0.01 compared to wild type; ^&P < 0.001 compared to DVB miniSOG; ^P < 0.01 compared to AVL miniSOG; ^$$P < 0.001 compared to nlp-40; ^$P < 0.01 compared to nlp-40; ^@P < 0.001 compared to wild type; n.s., not significant.

Fig. 2.

INX-1 is a gap junction protein that functionally couples AVL and DVB motor neurons at NMJs. (A) Representative images of AVL/DVB NMJs (arrowhead) and DVB soma (arrow) from young adults coexpressing INX-1::GFP and UNC-10/RIM1::mCherry fusion proteins in GABAergic motor neurons (AVL and DVB) under the unc-47 promoter. (B) Representative images of AVL/DVB NMJs (arrowhead) and DVB soma (arrow) from young adults coexpressing INX-1::GFP in AVL (under the unc-25(Δ) promoter) and INX-1::mCherry in DVB (under the flp-10 promoter). (C) Representative images of AVL/DVB NMJs (arrowhead) and DVB somas (arrow) from young adults coexpressing INX-1::GFP and mouse Cx36::mCherry fusion proteins under the unc-47 promoter. (D) Quantification of the number of Exp per defecation cycle in adults with the indicated genotypes. unc-33c cDNA was expressed under the GABAergic neuron-specific (Punc-47) promoter. “DVB HisCl” denotes expressing HisCl1 under the flp-10 promotor. (E) Quantification of the number of Exp per defecation cycle in adults with the indicated genotypes. Human PANX1 cDNA and synthetic/mouse Cx36 gene were expressed under the GABAergic neuron-specific (Punc-47) promoter. (Scale bar, 10 μm.) Asterisks indicate significant differences: ***P < 0.001 and *P < 0.05 in Student’s t test; n.s., not significant.

Fig. 3.

INX-1 synchronizes the activation of AVL and DVB motor neurons. (A) Representative images from videos showing GCaMP fluorescence in the AVL NMJ (arrowhead) and the DVB soma (arrow) before pBoc, at AVL activation onset, and at DVB activation onset in animals expressing GCaMP3 in DVB [under the unc-47(mini) promoter] and GCaMP6 in AVL (under the nmur-3 promoter). (Scale bar, 20 μm.) (B) Representative normalized traces showing the calcium dynamics at the AVL axon tip and DVB cell body during the defecation cycle in adult animals with the indicated genotypes. “same” denotes where the calcium spike in DVB cell body initiates within 500 ms after the calcium spike in AVL NMJ initiates; “AVL first” denotes where the calcium spike in DVB cell body initiates more than 500 ms after the calcium spike in AVL NMJ initiates, and “DVB first” denotes where the start of the calcium spike in DVB cell body precedes the start of calcium spike in AVL NMJ. (C) Calcium spike initiation time is quantified with the indicated genotypes. (D) Histogram showing the initiation time of each calcium spike in DVB cell body relative to the initiation of calcium spike in AVL NMJ with the indicated genotypes. (E) The frequency with which Exp occurs at the first or second spike is quantified for the indicated genotypes.

Fig. 4.

INX-1 promotes expulsion and DVB activation by AVL in response to pacemaker. (A) Schematic showing the strategy used to monitor DVB activation in the absence of pacemaker signaling. The NLP-40 receptor aex-2 was expressed only in the AVL motor neuron (under the flp-22 promoter), and the calcium indicator GCaMP3 was expressed in the DVB motor neuron (under the flp-10 promoter) in aex-2 mutants. (Scale bar, 20 μm.) (B) Quantification of the number of Exp per defecation cycle in adults with the indicated genotypes. “aex-2; AVL aex-2” denotes aex-2 cDNA expressed in AVL in aex-2 mutants. (C, Left) Representative images from a real time video showing GCaMP3 fluorescence in the NMJ (arrowhead) either right before the pBoc or the Exp step. (Scale bar, 20 μm.) (C, Right) Quantification of the number of calcium spikes observed during normal Exp. Means and SEs are shown. Asterisks indicate significant differences: *P < 0.05 in Student’s t test.

Fig. 5.

INX-1 inhibits ectopic activation of the DVB motor neuron. (A and C) Representative traces showing change of GCaMP3 fluorescence at the AVL/DVB NMJs during the defecation cycle in adult animals of the indicated genotypes expressing GCaMP3 under the unc-47(mini) promoter. (B and D, Left) Histograms showing the time when each calcium spike occurred after pBoc in wild-type and inx-1 mutants. (Right) Average frequency of calcium spikes per cycle grouped by normal and ectopic spikes in wild-type and inx-1 mutants. Means and SEs are shown. Asterisks indicate significant differences: ***P < 0.001 in Student’s t test.

Fig. 6.

Ectopic activation of the DVB motor neuron in inx-1 mutants are suppressed in egl-19 or egl-36(gf) mutants. (A) Diagram of the pacemaker controlled signaling pathway that leads to calcium influx at AVL/DVB NMJs and contraction of the enteric muscle (Exp). NP = neuropeptide; ACY = adenylate cyclase. (B) Quantification of the number of Exp per defecation cycle in adults of the indicated genotypes. PKA[DN] denotes a dominant negative PKA transgene expressed in GABAergic neurons (under the unc-47 promoter). (C) Average frequency of normally timed and ectopic (greater than 5 s) calcium spike per cycle in the indicated mutants. (D and E) Quantification of the number of Exp per defecation cycle in adults of the indicated genotypes. PKA[CA] denotes a constitutively active PKA transgene expressed in GABAergic neurons (under the unc-47 promoter). Neuronal egl-36(gf) denotes egl-36 (gain-of-function) transgene expressed under the GABAergic unc-47 promoter. (F) Average frequency of normally timed and ectopic (greater than 5 s) calcium spike per cycle in the indicated mutants. Means and SEs are shown. Asterisks indicate significant differences: ***P < 0.001 and **P < 0.01 in Student’s t test; n.s., not significant.

Fig. 7.

Working model for INX-1 in the expulsion step of the DMP. (Left) During the defecation cycle, calcium oscillation occurs in the intestine around every 50 s. SNT-2 on the DCVs senses the calcium and leads to NLP-40 release from the intestine. NLP-40 activates its receptor, AEX-2, which leads activation of ACY-1 to generate cAMP. Increased cAMP activates PKA, which results in calcium influx to GABAergic motor neurons (AVL and DVB), triggering GABA release. The released GABA activates its receptor, EXP-1, which leads to contraction of enteric muscles (Exp). During NLP-40–induced signaling, INX-1 promotes calcium influx in one neuron in response to activation of the other neuron, leading to synchronized GABA release from both NMJs. (Right) During cycle intervals, INX-1 suppresses ectopic activation of the motor neurons by inhibiting ectopic calcium transients. INX-1 sharpens NMJ output by mediating lateral excitation in the presence of signal and shunting inhibition in between cycles when signal is low. Dotted lines are experimentally not shown.