ACR-12 ionotropic acetylcholine receptor complexes regulate inhibitory motor neuron activity in Caenorhabditis elegans

Heterogeneity in the composition of neurotransmitter receptors is thought to provide functional diversity that may be important in patterning neural activity and shaping behavior (Dani and Bertrand, 2007; Sassoe-Pognetto, 2011). However, this idea has remained difficult to evaluate directly because of the complexity of neuronal connectivity patterns and uncertainty about the molecular composition of specific receptor types in vivo. Here we dissect how molecular diversity across receptor types contributes to the coordinated activity of excitatory and inhibitory motor neurons in the nematode Caenorhabditis elegans. We show that excitatory and inhibitory motor neurons express distinct populations of ionotropic acetylcholine receptors (iAChRs) requiring the ACR-12 subunit. The activity level of excitatory motor neurons is influenced through activation of nonsynaptic iAChRs (Jospin et al., 2009; Barbagallo et al., 2010). In contrast, synaptic coupling of excitatory and inhibitory motor neurons is achieved through a second population of iAChRs specifically localized at postsynaptic sites on inhibitory motor neurons. Loss of ACR-12 iAChRs from inhibitory motor neurons leads to reduced synaptic drive, decreased inhibitory neuromuscular signaling, and variability in the sinusoidal motor pattern. Our results provide new insights into mechanisms that establish appropriately balanced excitation and inhibition in the generation of a rhythmic motor behavior and reveal functionally diverse roles for iAChR-mediated signaling in this process

Monoaminergic Orchestration of Motor Programs in a Complex C. elegans Behavior

Monoamines provide chemical codes of behavioral states. However, the neural mechanisms of monoaminergic orchestration of behavior are poorly understood. Touch elicits an escape response in Caenorhabditis elegans where the animal moves backward and turns to change its direction of locomotion. We show that the tyramine receptor SER-2 acts through a $G\alpha_o$ pathway to inhibit neurotransmitter release from GABAergic motor neurons that synapse onto ventral body wall muscles. Extrasynaptic activation of SER-2 facilitates ventral body wall muscle contraction, contributing to the tight ventral turn that allows the animal to navigate away from a threatening stimulus. Tyramine temporally coordinates the different phases of the escape response through the synaptic activation of the fast-acting ionotropic receptor, LGC-55, and extrasynaptic activation of the slow-acting metabotropic receptor, SER-2. Our studies show, at the level of single cells, how a sensory input recruits the action of a monoamine to change neural circuit properties and orchestrate a compound motor sequence.Physic

Structural basis for the antiarrhythmic blockade of a potassium channel with a small molecule

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154620/1/fsb2fj201700349r.pd

Impaired dopamine release and synaptic plasticity in the striatum of parkin-/- mice

Parkin is the most common causative gene of juvenile and early-onset familial Parkinson's diseases and is thought to function as an E3 ubiquitin ligase in the ubiquitin-proteasome system. However, it remains unclear how loss of Parkin protein causes dopaminergic dysfunction and nigral neurodegeneration. To investigate the pathogenic mechanism underlying these mutations, we used parkin-/- mice to study its physiological function in the nigrostriatal circuit. Amperometric recordings showed decreases in evoked dopamine release in acute striatal slices of parkin-/- mice and reductions in the total catecholamine release and quantal size in dissociated chromaffin cells derived from parkin-/- mice. Intracellular recordings of striatal medium spiny neurons revealed impairments of long-term depression and long-term potentiation in parkin-/- mice, whereas long-term potentiation was normal in the Schaeffer collateral pathway of the hippocampus. Levels of dopamine receptors and dopamine transporters were normal in the parkin-/- striatum. These results indicate that Parkin is involved in the regulation of evoked dopamine release and striatal synaptic plasticity in the nigrostriatal pathway, and suggest that impairment in evoked dopamine release may represent a common pathophysiological change in recessive parkinsonism

Targeted disruption of glycogen synthase kinase-3β in cardiomyocytes attenuates cardiac parasympathetic dysfunction in type 1 diabetic Akita mice.

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3β, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3β, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3β might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3β. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3β. Using this mouse, we demonstrate that attenuation of GSK3β expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3β deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3β activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression

Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial parkinsonism-linked gene DJ-1

The manifestations of Parkinson's disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions

<i>ser-2</i> mutants are partially resistant to the paralytic effects of exogenous tyramine.

<p>(A) Tyramine induces immobilization in a dose-dependent manner. Shown is the percentage of animals immobilized after 10 min on agar plates supplemented with tyramine. Wild-type animals become fully immobilized at concentrations above 30 mM tyramine, while <i>ser-2</i> mutants continue sustained movement. Each data point represents the mean ± the standard error of the mean (SEM) for at least three trials, totaling a minimum of 30 animals. (B) G-protein signaling mutants are resistant to the paralytic of effects of exogenous tyramine. Shown is the percentage of animals that become immobilized after 10 min on 30 mM tyramine. Each bar represents the mean ± SEM for at least four trials totaling a minimum of 40 animals. (Inset) Schematic representation of the Gα_o and Gα_q signaling pathways that modulate locomotion in <i>C. elegans</i>. The genetic data suggest that SER-2 acts in the Gα_o pathway. The names of the human orthologs are shown. Rescue denotes the transgenic line <i>Pser-2</i>::SER-2; <i>ser-2(pk1357).</i> (C, D) Tyramine affects locomotion and head movements through different mechanisms. Shown is the percentage of animals with sustained body (C) or head (D) movements on 30 mM tyramine. <i>ser-2</i> mutants are partially resistant to the effects of tyramine on body movements, but not head movements. <i>lgc-55</i> mutants continue to move their heads through the duration of the assay. Each data point represents the mean percentage of animals that become immobilized by tyramine each minute for 20 min ± SEM for at least six trials, totaling a minimum of 60 animals. Head movements were analyzed in an <i>unc-3</i> mutant background. <i>unc-3</i> mutants make few body movements but display normal head movements, and are wild-type for the <i>ser-2 and lgc-55</i> loci. Statistical significance to wild-type: ***<i>p</i><0.0001, two-tailed Student's <i>t</i> test. See also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001529#pbio.1001529.s001" target="_blank">Figures S1</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001529#pbio.1001529.s002" target="_blank">S2</a>.</p

Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial parkinsonism-linked gene DJ-1

The manifestations of Parkinson's disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions

<i>ser-2</i> is expressed in a subset of GABAergic motor neurons.

<p>(A) A composite DIC image with fluorescent overlay showing that the <i>Pser</i>-2::mCherry transcriptional reporter is expressed in head muscles, head neurons, and neurons in the ventral nerve cord. (B–D) Transgenic animal showing coexpression of <i>Pser-2::</i>mCherry (B) and <i>Punc-47::</i>GFP, which labels all GABAergic motor neurons (C). <i>Pser-2::</i>mCherry is strongly expressed in the GABAergic VD neurons but not the DD neurons (D). Anterior is to the left. Scale bar is 20 µm. (E) Exogenous tyramine induces immobilization though the activation of SER-2 and Gα_o signaling pathway in the GABAergic neurons. Shown is the percentage of animals that become immobilized after 10 min on 30 mM tyramine. Loss-of-function of <i>unc-25</i> (glutamic acid decarboxylase) suppresses the tyramine resistance of <i>ser-2</i> mutant animals. <i>unc-25</i> (GABA deficient) mutants and <i>unc-25; ser-2(pk1357)</i> double mutants are not resistant to the paralytic effects of exogenous tyramine. Expression of SER-2 in all GABAergic neurons (<i>Punc-47</i>::SER-2) restores sensitivity of <i>ser-2</i> mutants to exogenous tyramine. Expression of GOA-1/Gα_o or EAT-16/RGS in all GABAergic neurons (<i>Punc-47</i>::GOA-1 or <i>Punc-47</i>::EAT-16) partially restores sensitivity to exogenous tyramine in the respective <i>goa-1</i> and <i>eat-16</i> mutants. Each bar represents the mean ± SEM for at least three trials, totaling a minimum of 30 animals.</p

<i>ser-2</i> mutants make shallow omega bends.

<p>(A) Distribution of touch-induced reversals ending in an omega turn. Omega turns are more likely to occur after longer reversals (>3 body bends). Wild-type and <i>ser-2</i> mutant animals initiate omega turns at the same rate (<i>n</i>≥150 per genotype). (B) Schematic representation of the omega angle. The omega angle was measured as the angle from the deepest point in the ventral bend to the closest points anterior and posterior of the animal. Images were adapted from movies of animals in the most ventrally contracted state of the escape response. (C) Percent of omega turns where the animal's nose touches the tail during the execution of the turn (closed omega turn). <i>ser-2</i> mutants [<i>ser-2(ok2103)</i>, <i>n</i> = 52; <i>ser-2(pk1357)</i>, <i>n</i> = 6 2] touch nose to tail less frequently than wild-type (<i>n</i> = 51) in omega turns induced by both touch (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001529#pbio.1001529.s010" target="_blank">Movies S4</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001529#pbio.1001529.s011" target="_blank">S5</a>) and blue light in a <i>Pmec-4</i>::ChR2 background [<i>Pmec-4</i>::ChR2, <i>n</i> = 38; <i>ser-2(ok2103)</i>; <i>Pmec-4</i>::ChR2, <i>n</i> = 43; <i>ser-2(pk1357)</i>; <i>Pmec-4</i>::ChR2, <i>n</i> = 28]. Tyramine/octopamine-deficient <i>tdc-1</i> mutants touch nose to tail less frequently than wild-type [<i>tdc-1(n3420)</i>, <i>n</i> = 144], while octopamine-deficient <i>tbh-1</i> mutants close omega turns like the wild-type [<i>tbh-1(n3247)</i>, <i>n</i> = 153]. Genomic rescue lines partially restore this omega turning defect (<i>ser-2</i> rescue line 2, <i>n</i> = 20; <i>ser-2</i> rescue line 3, <i>n</i> = 21). (D) Average omega angle measured after touch or exposure to blue light in a <i>Pmec-4</i>::ChR2 background [<i>Pmec-4</i>::ChR2, <i>n</i> = 38; <i>Pmec-4</i>::ChR2; <i>ser-2(ok2103)</i>, <i>n</i> = 43; <i>Pmec-4</i>::ChR2; <i>ser-2(pk2103)</i>, <i>n</i> =  28]. <i>ser-2</i> mutants [<i>ser-2(ok2103)</i>, <i>n = </i>52; <i>ser-2(pk1357)</i>, <i>n</i> = 62] and tyramine/octopamine-deficient mutants [<i>tdc-1(n3420)</i>, <i>n = </i>35] make a wider omega turn than wild-type (<i>n</i> = 51). Octopamine-deficient <i>tbh-1</i> mutants do not make wider omega turns [<i>tbh-1(n3247)</i>, <i>n</i> = 16]. Genomic rescue lines partially restore the omega angle defect of the mutants (<i>ser-2</i> rescue line 2, <i>n</i> = 20; <i>ser-2</i> rescue line 3, <i>n</i> = 21). (E) Escape angles were measured from the direction of the reversal (induced by gentle anterior touch) to the direction of reinitiated forward locomotion. (F) Distribution of escape angles. Dashed grey line indicates average. Wild-type animals and <i>tbh-1</i> mutants escape in the opposite direction from the touch stimulus [wt, 179°±5°, <i>n</i> = 42; <i>tbh-1(n3427)</i>, 177°±11°, <i>n</i> = 16]. <i>ser-2</i> mutants and <i>tdc-1</i> mutants make a shallower escape angle [<i>ser-2 (ok2103)</i>, 157.5°±5°, <i>n</i> = 53; <i>ser-2(pk1357)</i>, 150°±5°, <i>n</i> = 46; <i>tdc-1(n3420)</i>, 143.3°±6°, <i>n = </i>35]. Genomic rescue lines restore the escape angle to wild-type levels (<i>ser-2</i> rescue line 2, 173°±7°, <i>n</i> = 12; <i>ser-2</i> rescue line 3, 168.5°±5°, <i>n</i> = 20). Rescue denotes the transgenic line <i>Pser-2</i>::SER-2; <i>ser-2(pk1357).</i> Error bars depict SEM. Statistical differences calculated from wild-type unless otherwise indicated: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, two-tailed Student's <i>t</i> test.</p