alx , a Zebrafish Homolog of Chx10 , Marks Ipsilateral Descending Excitatory Interneurons That Participate in the Regulation of Spinal Locomotor Circuits

“For stochastic expression of monomeric red fluorescent protein (mRFP) (Campbell et al., 2002), a DNA construct in which mRFP (a gift from R. Tsien, University of California, San Diego, La Jolla, CA) was placed under a goldfish neural tubulin promoter (Hieber et al., 1998) was created and used.”

“A high-divalent cation concentration raises spike threshold (Frankenhaeuser and Hodgkin, 1957) and thereby reduces conduction in polysynaptic pathways.”

“To obtain isolated labeling in transgenic fish for morphological analysis, we used the Cre/loxP recombination system (Gu et al., 1994).”

“To generate alx reporter constructs, we followed the method by Lee et al. (2001), which uses BAC homologous recombination system (a gift from N. Copeland, National Cancer Institute, Frederick, MD).”

“En1 and Isl1 were used as markers for V1 neurons and motoneurons, respectively (Ericson et al., 1997; Briscoe et al., 2000 In addition to the dorsal cell marked by the arrow, most of the other GFP-positive cells (arrowheads) are also slightly shifted dorsally from the dorsal edge of the main Nkx6.”

“For the Kaede construct (Ando et al., 2002), the EGFP sequence in the template plasmid DNA was replaced by Kaede (a gift from A. Miyawaki, RIKEN, Saitama, Japan).”

“Studies in the cat have led to the classification of several spinal interneurons with roles in sensory/motor pathways (Jankowska, 1992).”

“Spinal motor circuits are best characterized in anamniotes such as lampreys and frog tadpoles, in which functional subtypes of interneurons are well identified (Roberts et al., 1998; Grillner, 2003).”

“To increase efficiency of transgenesis, we inserted an I-SceI meganuclease recognition site (Thermes et al., 2002) into the BAC DNA by homologous recombination.”

“Lhx3, in amniotes, has been shown to mark both V2 neurons and motoneurons (Ericson et al., 1997; Sharma et al., 1998; Thaler et al., 1999).”

“Microinjection of DNA into zebrafish embryos was performed as described previously (Higashijima et al., 1997) using BAC DNA at 40 –50 ng/␮l and plasmid DNA at 20 ng/␮l. BAC DNA was coinjected with I-SceI (Thermes et al., 2002).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“Recently, it has been shown that neurons that express the EphA4 tyrosine kinase receptor are important for mammalian locomotion (Kullander et al., 2003) and that a subset of these neurons excite motoneurons on the same side (Butt et al.”

“Consistent with this, Chx10-positive neurons in mice are labeled by the application of a dextran-conjugated dye caudal and ipsilateral to their somata in the spinal cord (Thaler et al., 1999).”

“Ipsilateral descending neurons having similar morphological features have been observed previously in zebrafish and named circumferential ipsilateral descending (CiD) neurons or ventral longitudinal descending neurons (Bernhardt et al., 1990; Kuwada et al., 1990; Hale et al., 2001).”

“These neurons are rhythmically active in phase with ventral root activity and make glutamatergic excitatory connections onto motoneurons (Dale and Roberts, 1985; Buchanan and Grillner, 1987).”

“The VIAAT protein is responsible for loading GABA and glycine into the synaptic vesicles of inhibitory neurons and is known to be expressed in GABAergic and glycinergic neurons (Sagne et al., 1997).”

“These neurons are rhythmically active in phase with ventral root activity and make glutamatergic excitatory connections onto motoneurons (Dale and Roberts, 1985; Buchanan and Grillner, 1987).”

“1 is also expressed in postmitotic neurons (Sander et al., 2000).”

“In amniotes, V1 neurons clearly show diversification and contain a minimum of two different cell types, Renshaw cells and Ia inhibitory neurons (Sapir et al., 2004; Alvarez et al., 2005).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“Knowledge of neuronal circuits responsible for locomotion has been further advanced in rodent preparations, in which, for example, several classes of commissural interneurons that are likely involved in locomotor control have been identified (Kiehn and Butt, 2003).”

“However, electrical coupling is also present in the adult CNS (Bennett et al., 1963; Ringham, 1975).”

“This notion comes from many lines of evidence including lesions, pharmacology, and paired recordings (Roberts et al., 1998; Buchanan, 2001).”

“Observations of live and fixed tissue (except for samples after electrophysiological recording) were performed essentially as described previously (Hale et al., 2001; Higashijima et al., 2004b,c).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“Single cell labeling with rhodamine– dextran by electroporation was performed essentially as described by Bhatt et al. (2004).”

“Many spinal neurons are electrically coupled at very early stages (Saint-Amant and Drapeau, 2001).”

“Extracellular recordings from motor nerves were performed as described by Masino and Fetcho (2005).”

“However, a calcium imaging study in zebrafish (Ritter et al., 2001) has shown that two different populations of interneurons, multipolar commissural descending (MCoD) neurons and CiD neurons, are differentially activated during two different behaviors (slow swimming and escape).”

“In goldfish, Mauthner neurons make monosynaptic excitatory connections to both motoneurons and ipsilateral descending neurons, which themselves make monosynaptic excitatory connections onto motoneurons (Fetcho, 1991 ).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“8 with NaOH (Drapeau et al., 1999).”

“This notion comes from many lines of evidence including lesions, pharmacology, and paired recordings (Roberts et al., 1998; Buchanan, 2001).”

“Cells that derive from a particular domain early in development could differ from those born at later stages (Marquardt and Pfaff, 2001).”

“One emerging view is that early V0 –V3 subdivisions of neurons in the embryonic spinal cord represent a primitive ground state that undergoes further diversification during development and/or evolution (Goulding et al., 2002; Goulding and Pfaff, 2005).”

“Soffe (1993) has shown that the same ipsilateral descending neurons are recruited both in swimming and struggling.”

“Ipsilateral descending neurons having similar morphological features have been observed previously in zebrafish and named circumferential ipsilateral descending (CiD) neurons or ventral longitudinal descending neurons (Bernhardt et al., 1990; Kuwada et al., 1990; Hale et al., 2001).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“In amniotes, V2 neurons are subdivided into at least Chx10-positive V2a and Gata2/3-positive V2b neurons (Karunaratne et al., 2002).”

“This point awaits the analysis of gata2/ 3-positive neurons in zebrafish (Neave et al., 1995).”

“…vertebrates and amniotes share common primary axonal trajectories , transmitter phenotype, and physiological roles in motor circuits (Saueressig et al., 1999; Wenner et al., 2000; Higashijima et al., 2004a; Li et al., 2004; Sapir et al., 2004; Goulding and Pfaff, 2005; Gosgnach et al., 2006).”

“An alx-positive bacterial artificial chromosome (BAC) was identified by blasting the alx cDNA sequence (Barabino et al., 1997) against the BAC sequences of the Sanger Institute (http://www.”

“Recently, it has been shown that neurons that express the EphA4 tyrosine kinase receptor are important for mammalian locomotion (Kullander et al., 2003) and that a subset of these neurons excite motoneurons on the same side (Butt et al., 2005).”

“However, electrical coupling is also present in the adult CNS (Bennett et al., 1963; Ringham, 1975).”

“A connection of the former is known in goldfish (Fetcho , 1992), but we are the first to describe the latter connection.”

“2008), Tg(vsx2:eGFP) (Kimura et al. 2006), Tg(pou4f3:mGFP) (Xiao et al.”

“The activities of these neurons during swimming have been studied by electrophysiological analysis of transgenic fish expressing GFP under the control of the En-1 and the alx promoter (26, 27).”