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1Howard Hughes Medical Institute and 2Department of Neurobiology and Behavior, State University of New York, Stony Brook; and 3Department of Neurobiology and Behavior, Cornell University, Ithaca, New York
Submitted 21 May 2008; accepted in final form 14 August 2008
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ABSTRACT |
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INTRODUCTION |
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; Eulenburg et al. 2005). GlyT1 is present in glia surrounding excitatory glutamatergic synapses in the brain (Smith et al. 1992) where glycine serves as a co-agonist for the N-methyl-D-aspartate (NMDA) receptor (Johnson and Ascher 1987). Signaling through NMDA receptors is augmented in mice with reduced GlyT1 function due to elevated levels of synaptic glycine (Gabernet et al. 2005; Martina et al. 2005; Tsai et al. 2004). GlyT1 is also present in glia surrounding inhibitory neurons, where the transporter controls levels of tonic inhibition in the hindbrain and spinal cord (Bradaia et al. 2004; Cui et al. 2005; Gomeza et al. 2003). In addition to setting levels of tonic inhibition, GlyT1 plays a modest role in shaping synaptic events: inactivation of GlyT1 either pharmacologically (Bradaia et al. 2004) or genetically (Gomeza et al. 2003), results in a slowing in decay kinetics with little change in peak amplitudes of spontaneous inhibitory synaptic currents. By controlling the concentration of CNS glycine, GlyT1 both modulates the amplitude of glutamatergic synaptic responses through the excitatory NMDA receptor and globally regulates levels of tonic inhibition through the inhibitory glycine receptor.
The zebrafish GlyT1 mutant shocked provides an alternate vertebrate genetic model in which to study the impact of GlyT1 on glycinergic signaling. In contrast to mouse glyT1 knockouts that die at birth, zebrafish GlyT1 mutants exhibit a dramatic behavioral recovery. Because the zebrafish preparation provides unprecedented access to nerve and muscle for recording (Luna and Brehm 2006; Wen and Brehm 2005), we were able to investigate the physiological basis for both the immotile and recovered mutant phenotypes. Mechanisms underlying the mutant phenotype had been the subject of controversy (Cui et al. 2004; Luna et al. 2004) as to whether muscle or CNS functional deficits were causal to the phenotype. Even after identification of the mutated gene (Cui et al. 2005), it was not known to what extent synaptic transmission was affected in zebrafish GlyT1 mutants, or the relationship between the initial motility dysfunction and later recovery of swimming behaviors. Our analyses of shocked mutant fish revealed dual roles for the glial glycine transporter. First, we found that GlyT1 plays a major role in shaping inhibitory, glycinergic synaptic transmission. Second, as shown for the mouse mutant, we found that GlyT1 participates in regulating global levels of glycine and confirmed that the initial behavioral dysfunction was due to an increase in tonic CNS inhibition. In addition, we identify homeostatic changes including increased CNS glycine tolerance, reduced glycinergic synaptic potentials and reduced glycine receptor expression that likely represent compensatory mechanisms linked to functional behavioral recovery.
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METHODS |
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For physiology, larval fish between the ages of 50 and 106 h post fertilization (hpf) were prepared as detailed elsewhere (Wen and Brehm 2005) with the following differences. To preserve the Mauthner neurons, fish were not decapitated. Following removal of the skin, they were paralyzed by application of 1 mg/ml 
-bungarotoxin for 5 min (Sigma, St. Louis MO). Subsequently, fish were treated with 1 mg/ml collagenase (Invitrogen, Carlsbad CA) to facilitate removal of the muscle cells overlaying the spinal cord. Collagenase treatment was followed by extensive washes in bath solution prior to recording. The bath recording solution contained (in mM) 140 NaCl, 10 Na-HEPES, 3 KCl, 2 CaCl2, 1 MgCl2, and 13 sorbitol, pH 7.4 and 293 mosM. Patch recording pipettes were filled with (in mM) 134 K-gluconate, 10 K-HEPES, 10 K-EGTA, and 6 KCl, pH 7.2 plus 0.03% sulforhodamine B (Sigma) for post recording verification of cell type. Extracellular stimulating pipettes were pulled to a long taper with a final tip size of 
2–3 µm, and filled with 140 mM NaCl. This pipette was placed adjacent to Mauthner ipsi- or contralateral axons in tail segments 12–14, and constant current pulses (200 µs, 2–5 µA) were delivered at 0.5 Hz (AM Systems Model 2100 stimulator).
Fish were prepared for immunohistochemical analysis as previously described (Ono et al. 2004). Anti-GlyR antibodies, (mAB4a: Synaptic Systems GmbH, Goettingen, Germany), were used at 1:100 dilution. After a 30-min incubation in 100 µg/mL RNase A, prepared fish were mounted in Vectashield with propidium iodide (PPI; Vector Labs, Burlingame, CA). and images were captured on a Zeiss 510 Meta confocal microscope. Image settings were first established using wild-type specimens and then applied to mutant image collection so staining could be directly compared. Zeiss LSM 510 image analysis software was used to quantify fluorescence intensity in each neuronal cell body. Single z-sections in which the nucleus was largest in diameter were selected for analysis. Fluorescence was then quantified in a line below the nucleus and the maximum value recorded for comparison across neurons in mutant and wild-type spinal cord.
Stocks of 10 mM N [3-(4'-fluorophenyl)-3-(4'phenylphenoxy) propyl]sarcosine (NFPS, Sigma) dissolved in DMSO were diluted in 10% Hank's balanced salt solution (HBSS, Invitrogen) just prior to use. Strychnine (Sigma) was freshly made as 100 mM stock in chloroform and diluted for use in 10% HBSS or bath solution. 2-amino-5-phosphovaleric acid (APV; Sigma) was made as 10 mM stock in water, and stored at –20°C. 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX; Sigma) was made as 10 mM stocks in water and stored in the dark at 4°C.
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RESULTS |
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). All three mutant lines exhibited severely compromised touch avoidance responses to mechanical stimulation during early development. The impact of the te301 mutation was assessed by characterizing touch avoidance of fish either homozygous or heterozygous for the shocked te301 allele at several developmental stages. To elicit an escape response, fish were tapped on the head or tail with a tungsten wire and the subsequent movement was filmed at 1,000 frame/s with a high-speed CCD camera. At 30 hpf, wild-type fish spontaneously generated a quick coiling response when dechorionated (Fig. 1A), whereas, in response to mechanical stimulation, homozygous shocked embryos were motionless (Fig. 1B). shocked embryos also spontaneously coil indicating that mutant muscle is contractile but only from 17 to 21 hpf and at a reduced frequency (Cui et al. 2005). At 56 hpf, wild-type fish generated a robust escape response, initiated by a C-bend and immediately followed by rhythmic swimming responses (Fig. 1A). Homozygous shocked generated a single protracted C-bend followed by an abrupt termination of swimming (Fig. 1B). Remarkably, during the subsequent 48 h of development (104 hpf), homozygote shocked mutants naturally acquired the ability to mount normal stimulus-evoked escape responses (Fig. 1B). Heterozygous shocked were indistinguishable from wild-type fish at all stages (Fig. 1C), so future reference to mutant fish applies exclusively to homozygotes.
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). The te301 shocked allele (Luna et al. 2004) was independently mapped to a region on linkage group 2 which contains the glyT1 gene. Consequently, we sequenced genomic glyT1 from te301 and identified a mutation at position 893 that converted the wild-type cysteine to a mutant tyrosine. This residue is located in the middle of the sixth transmembrane domain of the GlyT1 protein. dCAPS markers, used to track the mutation, segregated with the mutant phenotype 100% of the time (n = 500), indicating that the mutation is responsible for the motility dysfunction. Also, as shown for ta229g, morpholinos directed against GlyT1 phenocopied the shocked motility dysfunction in wild-type fish (Cui et al. 2005). For te301, two distinct morpholinos directed against the third and fourth intron/exon boundaries of the glyT1 gene independently mimicked early aspects of the shocked phenotype when injected into wild-type embryos at the one-to-two-cell stage (data not shown). Finally, all aspects of the shocked te301 phenotype including embryonic immotility at 30 hpf, the aborted escape response at 56 hpf, and subsequent behavioral recovery at 104 hpf were recapitulated in wild-type fish raised in 1 µM NFPS, a specific blocker of GlyT1 function (Fig. 1D) (Aubrey and Vandenberg 2001), indicating that GlyT1 mutations ta229g (Cui et al. 2005) and te301 both severely compromise GlyT1 function.
In a previous study, rhythmic motor drive recorded in muscle could be restored in zebrafish GlyT1 mutants by irrigating brain ventricles and relieving CNS glycine build-up (Cui et al. 2005). We used a variation of this technique to establish the glycine levels required for the behavioral phenotype of shocked te301. At the peak of the motility phenotype (48–72 hpf), mutants exhibit an exaggerated and prolonged C-bend, which abruptly terminates the subsequent rhythmic swimming (Fig. 1B). The wild-type escape behavior could be restored in GlyT1 mutants by removal of the skin over the fourth ventricle of the hindbrain in solution lacking glycine. Under these conditions, improved swimming was evident within 15–30 min after surgical manipulation in 81% of the fish tested (n = 21).
This technique was exploited to compare the sensitivity of the escape response to CNS glycine levels before and after phenotypic recovery in GlyT1 mutants. For this purpose, ventricle-exposed mutant and wild-type fish were allowed to swim in glycine-free solution to ensure normal activity following surgery. Embryos were then transferred to solutions containing 10 mM glycine, a concentration that consistently resulted in an aborted escape response in all fish tested, including wild type. Subsequently, individual GlyT1 mutant fish were exposed to solutions containing successively lower concentrations of glycine and tested for their ability to mount a wild-type escape response. Dose-response curves were constructed reflecting the percentage of fish tested at each glycine concentration that mounted a wild-type escape response. A linear fit of the log values of glycine concentration curves yielded the half-maximal concentration of glycine permissive to wild-type escapes (glycine tolerance). Comparisons at 50–58 hpf indicated a 186-fold difference in glycine tolerance between GlyT1 mutant and wild-type fish (Fig. 2A; sho 5.9 µM; n = 17; WT 1.1 mM; n = 13). However, at later developmental stages, when GlyT1 mutant fish naturally recovered the ability to mount a normal escape response, they exhibit a 61-fold increase in glycine tolerance (Fig. 2B; sho 360 µM; n = 30). Over this same period, wild-type glycine tolerance increased 3.5-fold (Fig. 2B; WT 3.9 mM; n = 15). An increased glycine tolerance was also obtained in wild-type fish that were reared in 1 µM NFPS. Prior to recovery and at the peak of the swimming dysfunction the half-maximal value for NFPS treated wild-type fish was 8.6 µM glycine (Fig. 2A, n = 7), and following behavioral recovery the value rose to 223.6 µM glycine (Fig. 2B, n = 10).
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). The two Mauthner cell bodies in the hindbrain extend axons that cross in the brain and descend the length of the spinal cord, forming excitatory synapses on ipsilateral primary motor neurons (Fig. 3A). Mauthner axons are also electrically coupled to glycinergic commissural interneurons that cross the spinal cord to inhibit contralateral primary motor neurons (Fetcho 1990). Escape responses were elicited by antidromic stimulation of the Mauthner axon while patch clamping a primary motor neuron. When the contralateral Mauthner axon was stimulated, a rapid hyperpolarization from –40 mV was followed by a slow depolarization in the motor neuron (Fig. 3A, Contra). In contrast, when the ipsilateral axon was stimulated, the response in the motor neuron was purely positive going and sufficiently large to result in the generation of an action potential (Fig. 3A, Ipsi). The ability of the Mauthner neuron to excite or inhibit motor neurons in GlyT1 mutant fish is consistent with the behavioral C-bend that occurs in response to touch (Fig. 1B).
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Taking advantage of the ability to isolate the glycinergic response by holding the membrane potential of the motor neuron at 0 mV, recordings of synaptic responses from GlyT1 mutant larvae at 50–58 hpf were compared with those of wild type. Representative synaptic responses recorded at 0 mV illustrated the larger amplitude and prolonged time course in GlyT1 mutant fish when compared with wild-type fish of the same age (Fig. 5A). The increased amplitude of GlyT1 mutant synaptic potentials was consistent at all membrane potentials tested (Fig. 5B). Overall comparisons indicated that the peak amplitude at 0 mV was significantly larger (P < 0.05) in GlyT1 mutants (26.7 ± 6.2 mV; n = 5) compared with wild type (15.0 ± 5.6 mV; n = 4). To quantify the overall response of the evoked inhibitory postsynaptic potentials (IPSPs), the area was integrated and plotted as a function of potential (Fig. 5C). The integral of the IPSP measured 876.7 ± 665.9 mV·ms (n = 5) in GlyT1 mutants compared with 194.8 ± 166.3 mV·ms (n = 4) for wild-type fish (P < 0.07). These differences in IPSP amplitude and kinetics did not reflect differences in motor neuron input resistance. Steady state measurements of input resistance between –80 and –30 mV indicated no significant differences (P = 0.14) between GlyT1 mutant (428.2 ± 81.1 M
) and wild-type fish (312.6 ± 107.8 M; Fig. 5D).
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) and recovered GlyT1 mutants (204.33 ± 39.5 M; Fig. 5H). Moreover, at the time corresponding to recovery, the input resistance for GlyT1 mutants was higher than wild-type fish.
To identify genes the differential expression of which could explain functional recovery in mutants, transcript levels were compared in wild-type and GlyT1 mutant fish. We performed a developmental time course for GlyT1 mRNA as well as for neuronal glycine transporter (GlyT2), NMDA receptor glycine binding subunit (NR1.1), and the alpha 1 subunit of the glycine receptor (GlyR1) (Fig. 6, A–D). The developmental profiles of GlyT1 mRNA in mutant and wild-type fish were similar. In contrast, at 50 hpf, GlyT2, NR1.1, and GlyR1 transcript levels in GlyT1 mutants were all significantly different from wild-type (Fig. 6, B–D). NR1.1 was slightly elevated while GlyR1 and GlyT2 were reduced in the mutants. At 122 hpf, when GlyT1 mutants had recovered the ability to swim, expression levels of GlyT2 and NR1.1 were similar to those in wild-type but expression levels of GlyR1 mRNA remained significantly reduced (P < 0.05; Fig. 6D, 122 hpf; WT 4.67 ± 0.43; n = 3; sho 2.33 ± 0.9; n = 3 independent RNA samples, 20 fish each).
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). In whole-mount immunohistochemistry of wild-type fish, the lateral spinal cord was heavily labeled as previously observed (McDearmid et al. 2006). To look more specifically at primary motor neurons, the mAB4a fluorescence was quantified in a subset of superficial neurons with large nuclei that reside in the lateral spinal cord (Fig. 6E). This class of cells should include the primary motor neurons. To control for consistency across samples, an independent fluorescent nuclear label, propidium iodide (PPI), was also quantified. At 50 hpf, when GlyT1 mutants are unable to swim, there were no significant differences between mutants and wild-type fish in peak fluorescence staining intensities for either mAB4a (P = 0.66; sho 3831 ± 241, n = 20; WT 3789 ± 353, n = 20) or PPI (P = 0.44; sho 2539 ± 390, n = 20; WT 2575 ± 253, n = 20). However, at 122 hpf following behavioral recovery, while PPI fluorescence remained unchanged (PPI: P = 0.23; sho 2835 ± 504, n = 20; WT 2528 ± 860, n = 20), there was significantly less GlyR protein in GlyT1 mutants (mAB4a: P < 0.05; sho 3140 ± 778, n = 20; WT 4064.35 ± 80, n = 20). |
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DISCUSSION |
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) of which ta229g (Cui et al. 2005) and te301 (present study) are known to represent point mutations in the glyT1 gene. GlyT1, a member of the SLC6 family of amino acid transporters, is responsible for glycine reuptake into glial cells (Guastella et al. 1992; Liu et al. 1992; Smith et al. 1992). In the te301 allele, a point mutation in the sixth transmembrane region (TM6) converts a cysteine to a tyrosine (C298Y). While the GlyT1 cysteine 298 is conserved across taxa, it is not conserved in other members of the SLC6 transporter family. In the bacterial SLC6 transporter LeuT, where crystal structure is known (Yamashita et al. 2005), TM6 has been implicated in substrate binding. Although the extent to which glycine transport is compromised in te301 is not known, the ta229g allele lacks the ability to transport glycine altogether (Cui et al. 2005), and both mutant alleles share the same motility defects. Moreover, shocked motility defects are phenocopied in wild-type fish by injecting morpholinos directed against GlyT1 transcript or by treatment with an inhibitor of GlyT1 function. These data support that the te301 GlyT1 mutation severely compromises glycine transporter function.
Our direct recordings of a CNS glycinergic synapse in shocked suggest that GlyT1 normally functions to shape fast glycinergic synaptic responses. At 50–58 hpf, inhibitory synaptic potentials in GlyT1 mutants were 50% larger in amplitude and 450% larger in area than wild-type. A role for glial transporters in synaptic function is well established at glutamatergic nerve terminals (Tzingounis and Wadiche 2007). Given the intimate relationship between nerve and glia required for involvement of a glial transporter in synaptic transmission, it would be expected to find variability in the extent to which the transporter regulates kinetics at different glycinergic synapses. Indeed the synapses examined on goldfish mauthner neurons (Titmus et al. 1996), mouse hypoglossal neurons (Gomeza et al. 2003), and rat lamina X spinal neurons (Bradaia et al. 2004) did not reveal a prominent role for GlyT1 in fast inhibitory transmission. Nonetheless, fast synaptic transmission was profoundly affected by the GlyT1 mutation at the glycinergic synapse onto the zebrafish primary motor neuron.
Our experimental findings provide further support for the role of GlyT1 in global glycinergic inhibition. The idea that glial glycine transporters regulate global glycine concentrations originated from mouse knockout models of the glyT1 gene (Gomeza et al. 2003). Recordings of respiratory circuit activity from the brain slices of GlyT1–/– newborn mice showed a significant reduction in the frequency of neuronal firing. Hypoglossal motor neurons exhibited increased membrane noise and standing current, both of which were strychnine-sensitive (Gomeza et al. 2003). Elegant studies in the zebrafish GlyT1 mutant ta229g also emphasize the importance of GlyT1 in setting global levels of glycine (Cui et al. 2005). Muscle recordings demonstrate that rhythmic motor output could be restored in GlyT1 mutants by irrigating the fourth ventricle of the brain with glycine-free solution (Cui et al. 2005). We adapted this technique by irrigating mutant brains with glycine free solution and monitoring recovery at the level of behavior. Reintroduction of glycine to the bath restored the GlyT1 mutant phenotype provided that the concentration was sufficiently high. These studies confirm that GlyT1 regulation of global CNS glycine levels is crucial for normal function of the motor circuit.
Exposing GlyT1 mutant and wild-type brains to known glycine concentrations allowed us both to titrate glycine concentrations permissive for normal behavior and to provide insight into the behavioral recovery that occurs in GlyT1 mutant fish during development. At 50–58 hpf, wild-type glycine tolerance was nearly 200-fold greater than in GlyT1 mutants. However, between 96 and 120 hpf, corresponding to the time that GlyT1 mutant fish acquired the ability to swim, glycine tolerance in mutants increased >60-fold. Over the same time period, the glycine tolerance in wild-type increased only threefold. Consequently, following the developmental acquisition of swimming by GlyT1 mutants, CNS glycine tolerance in mutants approaches that of wild-type fish.
What might account for the altered sensitivity to global glycine during development? Insights to possible mechanisms came from our recordings of inhibitory synaptic potentials from primary motor neurons. During the period of behavioral recovery in GlyT1 mutant fish, motor neuron inhibitory synaptic potentials decreased significantly, adopting the kinetics and amplitude of wild-type responses. The decreased response was not a secondary consequence of disproportionate changes in input resistance. Although the synaptic changes at this motor neuron synapse cannot account for the recovery, we suggest that this synapse serves to reflect synaptic changes that are taking place globally and that underlie increased glycine tolerance in recovered GlyT1 mutants.
We propose that the developmental decrease in synaptic potential amplitude results, in part, from a reduction in the number of postsynaptic glycine receptors. This is based on quantitative measurements of RNA coding for the alpha subunit of the glycine receptor as well as immunohistochemical labeling of motor neurons by anti-GlyR subunit antibodies. Because we detect a reduction in GlyR1 transcript levels in RNA samples isolated from whole animals, this reduction must have occurred throughout the nervous system. Indeed GlyR immunoreactivity is reduced in neurons viewed throughout the spinal cord. The largest decreases in glycine receptor expression measured in the motor neurons are only apparent after decreases in synaptic potentials. This could reflect the greater sensitivity of the physiological assays or that receptors are functionally inactivated prior to their transcriptional down-regulation. At early stages of excess inhibition, bath application of strychnine to block glycine receptors rescues rhythmic swimming (Supplemental Movie).1 (Cui et al. 2005). These findings support that reducing glycine receptor function is a viable mechanism for restoring swim circuit function.
By analogy to cholinergic synapses, a reduction in glycine receptor number would be expected to both reduce amplitude and accelerate kinetics of inhibitory responses in the absence of glycine uptake. At neuromuscular synapses, inhibition of acetylcholine hydrolysis increases the amplitude and time course of synaptic currents, much like that seen at inhibitory synapses of GlyT1 mutant fish before recovery. Experimental reduction of receptor density by application of either curare or alpha-bungarotoxin decreased the amplitude and greatly accelerated the kinetics of synaptic current (Katz and Miledi 1973). The proposed mechanism responsible for the altered kinetics is reduced probability of rebinding to receptors due to lowered density, thereby facilitating clearance from the synaptic cleft. Should similar mechanisms occur in the hindbrain, the altered density of glycine receptors would explain the observed homeostatic decrease in sensitivity to glycine.
Homeostatic changes in receptor expression have been described in other SLC6 transporter mutants including serotonin, norepinephrine, GABA, and dopamine transporter knockout mice (Bengel et al. 1997; Jensen et al. 2003; Jones et al. 1998; Xu et al. 2000). However, GlyT1–/– mice lack the compensatory changes in inhibitory synaptic machinery, including the mouse GlyR protein (Gomeza et al. 2003). This difference is likely due to the fact that GlyT1–/– mutant mice die at birth, whereas all other transporter mutant lines are viable and fertile, often studied days to weeks after birth (Bengel et al. 1998; Jensen et al. 2003; Jones et al. 1998; Xu et al. 2000). The compensatory downregulation of inhibitory glycine receptors in GlyT1 mutant zebrafish is likely to represent general homeostatic mechanisms that compensate for excessive levels of neurotransmitter when transporters are inactivated.
Compensatory receptor expression in response to changes in circuit-wide activity has been demonstrated at other vertebrate CNS synapses (reviewed in Turrigiano 2007); however, the signals that trigger the compensatory response are unknown. Because glycine levels produce tonic inhibition, it is likely that activity-dependent mechanisms are involved. This idea is supported by the fact that fast skeletal muscle exhibits electrical coupling in GlyT1 mutant larvae at a stage when wild-type fast muscle has already lost gap junctional coupling (Luna et al. 2004). Loss of electrical coupling in skeletal muscle also occurs in Xenopus and in this preparation has been shown to depend on CNS electrical activity (Armstrong et al. 1983). The mechanisms that trigger these dramatic homeostatic modifications in the GlyT1 mutant will be the focus of future studies.
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GRANTS |
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ACKNOWLEDGMENTS |
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Present addresses: G. Mandel, P. Brehm, and R. Mongeon, Vollum Institute, Oregon Health Science University, Portland, OR 97239; M. R. Gleason, Laboratory of Sensory Neuroscience, The Rockefeller University, 1230 York Ave., Box 314, New York, NY 10021; M. A. Masino, Dept. of Neuroscience, University of Minnesota, 321 Church St., 6-145 Jackson Hall, Minneapolis, MN 55455.
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FOOTNOTES |
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1 The online version of this article contains supplemental data. 
Present address and address for reprint requests and other correspondence: J. E. Dallman, Dept. of Biology, 1301 Memorial Dr., University of Miami, Coral Gables, FL, 33124 (E-mail: jdallman{at}bio.miami.edu)
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ABSTRACT
INTRODUCTION
, shocked mutants; 
, wild-type reared in 1 µM NFPS; and 
, wild-type control. The NFPS was included at all glycine concentrations during behavioral testing. The lines were fitted through 10–90% response curves by linear regression, and the 50% intersection of the line was used as a measure of sensitivity to glycine. At early stages [A, 50–58 h post fertilization (hpf)], GlyT1 mutants (n = 17) and wild-type fish in NFPS (n = 14) are nearly 200 times less glycine tolerant than wild-type fish (n = 13). After they regain normal motility (B, 98–122 hpf), GlyT1 mutants (n = 30) and wild-type fish in NFPS (n = 15) are only 11 times less glycine tolerant than wild-type fish.
) the axons of which cross the spinal cord to inhibit Ps on the opposite side. Axons of the Ps exit the spinal cord to innervate peripheral fast muscle (hatched parallelograms). SIpsi: stimulation of the ipsilateral Mauthner axon triggers an action potential. SContra: stimulation of the contralateral Mauthner axon triggers an inhibitory postsynaptic potential (IPSP) followed by a small depolarization. B: the relative amplitudes of the individual excitatory and inhibitory components of the synaptic potentials are dependent on membrane potential of the motor neuron. Synaptic potentials were triggered by stimulating the contralateral Mauthner axon in 50–58 hpf wild-type fish. Representative individual sweeps taken between –80 and 10 mV are shown. Near –40 mV the IPSP is negative-going and the excitatory postsynaptic potential (EPSP) is sufficiently positive to generate an action potential. Near –80 mV, both components are positive-going.
