Genes, Brain and Behavior (2014) 13: 633–642
doi: 10.1111/gbb.12153
Food deprivation and nicotine correct akinesia and freezing in Na+-leak current channel (NALCN)-deficient strains of Caenorhabditis elegans K. Bonnett† , R. Zweig‡ , E. J. Aamodt§ and D. S. Dwyer†,¶,∗ † Department
of Pharmacology, Toxicology and Neuroscience, of Neurology, § Department of Biochemistry and Molecular Biology, and ¶ Department of Psychiatry, LSU Health Sciences Center, Shreveport, LA, USA *Corresponding author: D. S. Dwyer, Department of Psychiatry, LSU Health Sciences Center, 1501 Kings Highway, Shreveport, LA 71130 USA. E-mail:
[email protected]
‡ Department
Mutations in various genes adversely affect locomotion in model organisms, and thus provide valuable clues about the complex processes that control movement. In Caenorhabditis elegans, loss-of-function mutations in the Na+ leak current channel (NALCN) and associated proteins (UNC-79 and UNC-80) cause akinesia and fainting (abrupt freezing of movement during escape from touch). It is not known how defects in the NALCN induce these phenotypes or if they are chronic and irreversible. Here, we report that akinesia and freezing are state-dependent and reversible in NALCN-deficient mutants (nca-1;nca-2, unc-79 and unc-80) when additional cation channels substitute for this protein. Two main measures of locomotion were evaluated: spontaneous movement (traversal of >2 head lengths during a 5 second observation period) and the touch-freeze response (movement greater than three body bends in response to tail touch). Food deprivation for as little as 3 min stimulated spontaneous movement and corrected the touch-freeze response. Conversely, food-deprived animals that moved normally in the absence of bacteria rapidly reverted to uncoordinated movement when re-exposed to food. The effects of food deprivation were mimicked by nicotine, which suggested that acetylcholine mediated the response. Nicotine appeared to act on interneurons or motor neurons rather than directly at the neuromuscular junction because levamisole, which stimulates muscle contraction, did not correct movement. Neural circuits have been proposed to account for the effects of food deprivation and nicotine on spontaneous movement and freezing. The NALCN may play an unrecognized role in human movement disorders characterized by akinesia and freezing gait. Keywords: Acetylcholine, akinesia, C. elegans, food deprivation, freezing gait, locomotion, neural circuit, nicotine, Parkinson’s disease, sodium leak current channel Received 3 April 2014, revised 16 June 2014, accepted for publication 26 June 2014
Combined genetic and behavioral analyzes in Caenorhabditis elegans have revealed numerous genes that adversely affect movement causing an uncoordinated or Unc phenotype (Brenner 1974). Mutations in more than 100 genes produce this phenotype, although the exact genetic defects responsible have not been identified in all cases. The Unc strains exhibit a wide array of locomotion defects, including coiling, kinked movements, akinetic states, dominant reversals and fainting (Brenner 1974; Wood 1988). Consequently, C. elegans represents a very attractive model system for learning about the regulation of motor behavior. Interestingly, some of the locomotion defects in C. elegans resemble the symptoms observed in human movement disorders. For example, strains with loss-of-function (lf) mutations in unc-2, which is a P/Q-type Ca++ channel (CACNA1A), show sluggish, kinked movements reminiscent of episodic ataxia type 2 caused by functional defects in the corresponding human gene (Jen et al. 2007). Similarly, unc-79(lf) and unc-80(lf) mutants show little spontaneous movement and freeze (faint) during escape from tail touch (Sedensky & Meneely 1987), which mimics the akinesia and frozen gait seen in patients with movement disorders such as Parkinson’s disease, primary progressive freezing gait and progressive supranuclear palsy (Factor 2008). The precise function of UNC-79 and UNC-80 is unknown, although they are required for the proper expression of the Na+ leak-current channel (NALCN; NCA-1 and NCA-2 in C. elegans) (Humphrey et al. 2007; Yeh et al. 2008). The NALCN is a leaky cation channel that brings the membrane potential closer to the threshold for depolarization, which facilitates activation of voltage-gated ion channels. It is involved in the regulation of rhythmical activity such as respiration (Lu et al. 2007), locomotion (Nash et al. 2002) and pacemaker activity in central pattern generator neurons (Lu & Feng 2011). In addition, the NALCN is activated by muscarinic cholinergic receptors in pancreatic 𝛽-cells (Swayne et al. 2009) where acetylcholine stimulates insulin release (Sharp et al. 1974). Knockout of NALCN reduces synaptic transmission by decreasing Ca++ transients (Yeh et al. 2008) suggesting that it normally enhances Ca++ influx during signaling. This effect may explain how NALCN helps establish synchronous firing of neurons by creating a balance between Ca++ -mediated depolarization and repolarization via Ca++ -activated K+ channels, and how it might regulate insulin secretion. Previously, we discovered that the NALCN is involved in the recovery of pharyngeal pumping in response to food deprivation, which we attributed to its effects on insulin secretion (Dwyer & Aamodt 2013). During those studies, we noticed that NALCN-deficient strains (nca-1;nca-2, unc-79 and unc-80) exposed to overnight starvation showed
© 2014 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society
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surprising and dramatic improvements in locomotion. To study this phenomenon in greater detail, we have evaluated the effects of long- (24 h) vs. short-term (3 body bends in response to touch, they were considered to be moving;
