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1Department of Physiology and Center for Neuroscience, University of Wisconsin Medical School, Madison, Wisconsin; and 2Division of Neuroscience, John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia
Submitted 25 February 2005; accepted in final form 3 May 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Cohen and Salzberg 1978; Grinvald et al. 1988; Momose-Sato et al. 2001; Wu et al. 1998). Images of fast voltage-sensitive dyes have provided valuable information about the functional organization of neural networks in a wide variety of preparations ranging from the isolated invertebrate cultured neurons (e.g., Parsons et al. 1991) to in vivo imaging of neuronal activity in the rat barrel cortex (Petersen et al. 2003).
Multiple-site optical recordings have also proven useful in studies mapping the spatial extent of neuronal excitation in tissues that are inaccessible to conventional electrophysiological recordings (reviewed by Salzberg 1985; Grinvald 1985). Voltage images have been fundamental to our understanding of the ontogeny of electrophysiological function and network organization in embryonic chick brain stem and spinal cord at stages when electrophysiological recordings are difficult, if not impossible (Arai et al. 1999; Mochida et al. 2001; Momose-Sato et al. 2003; Sato et al. 1998; reviewed by Momose-Sato et al. 2001). High temporal resolution optical measurements are advantageous in developing neuronal systems, because in the absence of myelin, the translucent tissue provides relatively large optical signals. In embryonic chick spinal cord, sensory stimulation triggered voltage images consisting of fast and slow signals. Based on pharmacological manipulations, it has been proposed that those were presynaptic action potentials and postsynaptic potentials, respectively. However, data were cautiously interpreted in the absence of quantitative comparison of membrane potentials and their respective optical signals. Combined electrophysiological and optical recordings revealed closely matched voltage images and potential changes in response to sensory and antidromic stimuli in chick spinal motoneurons (Wenner et al. 1996). Sophisticated combination of voltage-sensitive dye images and whole cell potential recordings in the rat barrel cortex in vivo showed a strong correlation between sensory-evoked subthreshold excitatory potentials and optical signals (Petersen et al. 2003).
Relatively little is known about the spatiotemporal pattern of sensory-evoked network activity in the rodent spinal cord. We have examined the spatiotemporal dynamics of spontaneous depolarizations in transverse slices of embryonic and newborn rat spinal cords using a voltage-sensitive dye with a 464-element photodiode-fiber optic camera (Demir et al. 2002). Real-time fluorescence images revealed coordinated rhythmic activity with spontaneous voltage oscillations alternating between mirror locations in the right and left sides of the cord. Extracellular potentials confirmed that these were representatives of local voltage changes.
The objectives of this study were 1) to characterize the spatiotemporal pattern of dorsal root–evoked network excitation using images from voltage-sensitive absorption dye, 2) to examine the correlation between whole cell potentials and simultaneously recorded local voltage images originated from neuronal ensembles in the area around the soma, and 3) to study the contribution of excitatory and inhibitory synapses to the spatiotemporal dynamics of evoked network activity.
A preliminary report of this study was published in an abstract form (Ziskind-Conhaim and Redman 2003).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Lumbar segments of the spinal cord were isolated from 1- to 4-day-old postnatal Sprague-Dawley rats (P1–P4). The procedure for dissecting out the spinal cord was similar to that described previously (Gao and Ziskind-Conhaim 1998). Postnatal rats were anesthetized by hypothermia, and the lumbar region of the spinal cord was removed and placed in oxygenated cold dissection solution. The isolated spinal cord was embedded in agar (2% in extracellular solution), and 400- to 600-µm transverse slices with dorsal roots intact were cut with a Leica VT1000S vibratome. Slices were incubated in extracellular solution at room temperature for 30–60 min.
Voltage-sensitive dye staining
Slices were stained for 30–40 min in extracellular solution containing the voltage-sensitive oxonol dye, RH-155 (0.2 mg/ml; Molecular Probes, Eugene, OR). The excess dye was washed away with dye-free extracellular solution. For comparison, in a few experiments, slices were stained with RH-482 (0.2–0.3 mg/ml; NK3630, Hayashibara Biochemical Laboratory, Okayama, Japan). RH-155 was the dye of choice in our experiments for two main reasons. First, slices incubated in RH-482 seemed to degenerate more rapidly, possibly because RH-482-containing solution foamed when aerated, making it difficult to adequately equilibrate it with 95% O2-5% CO2. Second, RH-155 had a larger signal-to-noise ratio than RH-482 (also Momose-Sato et al. 1999), although this might be partly attributed to its staining of glial cells (Kojima et al. 1999). Phototoxicity could not be detected with either dye (Chang and Jackson 2003). The disadvantage of staining with RH-155 was its relatively rapid washout during long-term recordings. One-hour superfusion in extracellular solution resulted in 
20% signal attenuation compared with <10% reduction in slices stained with RH-482. Similar dye characteristics were described previously during long-term recordings in hippocampal slices (Momose-Sato et al. 1999).
Voltage-sensitive dye imaging
Stained slices were transferred to a recording chamber mounted on the stage of an Olympus microscope (BX50WI) and were held with a nylon mesh in a glass-covered recording chamber. The slices were superfused with extracellular solution at a rate of 1–2 ml/min at a temperature of 29–31°C (Demir et al. 2002). Dorsal roots were stimulated using tight suction electrodes or fine tungsten wires insulated to about 10–20 µm from the tip. In most slices, square pulses of 300–800 µA/0.3–0.5 ms triggered optical responses in the ventral horn. Experiments were discontinued if stimuli did not produce optical signals in the ventral horn.
Light from a 100-W halogen bulb powered by a regulated power supply (Kepco, Flushing, NY) was passed through an interference filter with a transmission maximum at 710 ± 10 nm (Omega Optical, Brattleboro, VT). The filter was placed between the light source and the recording chamber (Fig. 1). The experimental approach was to detect changes in the intensity of transmitted light, which is inversely related to changes in light absorption. The light was focused onto a photodiode-fiber optic camera with 464 hexagonally arranged detectors (RedShirtImaging, Fairfield, CT), and voltage-sensitive dye signals were recorded with either a x10 (N.A. 0.4) or x20 (N.A. 0.95) objective. The diameter of the optical field of each photodiode was 30 µm at x20. The amplitude of optical signals was measured as peak change in absorption normalized to the resting light from each photodiode (
I/I).
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Whole cell voltage recording
To identify the photodiode corresponding to the site of whole cell voltage recording, digital images of the slice were first taken with a x10 objective, and those were overlaid on the image composed by the optic camera (Fig. 2, A and C). At the end of the experiment, a digital image was taken again with the recording pipette in place. Whole cell voltage recordings corresponded to optical signals detected by the photodiode in the center of the 464 hexagonally arranged detectors (Fig. 2C, x20). Neurons at specific locations in the transverse slice were visualized for whole cell recordings using infrared differential interference contrast videomicroscopy (Ziskind-Conhaim et al. 2003). The interference filter was removed, and the light was directed to the camera (Fig. 1). After the whole cell current-clamp configuration was established, the filter was reinserted for optical measurements.
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using a multi-stage puller (Sutter Instruments, Novato, CA). Intracellular potentials were recorded with an Axoclamp 2B amplifier (Axon Instruments, Union City, CA). Potentials were filtered at 3 kHz and digitized at 10–20 kHz. Membrane potentials were corrected off-line for a 10-mV liquid junction potential (Gao et al. 2001). Solutions
Dissection solution contained (in mM) 113 NaCl, 3 KCl, 1 CaCl2, 6 MgCl2, 25 NaHCO3, 1 NaH2PO4, and 11 glucose. The pH was 7.2–7.3, and the osmolarity was 300–310 mOsm. The extracellular solution was composed of (in mM) 113 NaCl, 3 KCl, 2 CaCl2, 1 MgCl2, 25 NaHCO3, 1 NaH2PO4, and 11 glucose. The solution was equilibrated with 95% O2-5% CO2 (pH 7.2 at 20–22°C). Patch pipettes were filled with solution containing (in mM) 140 K-Gluconate, 9 KCl, 10 HEPES, 0.2 EGTA, 1 Mg-ATP, and 0.1 GTP. The solution was adjusted to pH 7.2 using KOH, and the osmolarity was 290 mOsm.
Receptor antagonists used in our study included D-2-amino-5-phosphonovaleric acid (D-APV; 20 µM) and 6-cyano-7-nitroquinoxaline (CNQX; 10 µM), which are N-methyl-D-aspartate (NMDA) and non-NMDA receptor antagonists, respectively; strychnine (0.5 µM), a glycine receptor antagonist; and picrotoxin (10 µM), bicuculline (5 µM), and SR-95531 (1 µM), which are all GABAA receptor antagonists.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Typically, dorsal root stimulation evoked optical signals that propagated throughout the slice (Fig. 2, B and C). In most experiments, initial voltage images were captured with a x10 objective, because at this magnification, the 464-photodiode fiber optic camera detected voltage-dependent light changes from one entire side of the cord (Fig. 2, A and B). After confirming that dorsal root stimulation produced optical responses in the ventral horn, the x10 objective was replaced with a x20 objective to visualize neurons for whole cell recordings. The spatial resolution was higher with the x20 objective, because at this magnification, each photodiode recorded voltage-dependent light changes from one quarter of the area recorded using a x10 objective. However, under our experimental conditions, signals with a similar time-course were recorded with either objective (Figs. 2C and 3). This might imply that dorsal root stimulation produced a relatively homogeneous response in local neuronal ensembles.
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) that might be associated with electrical activity in neural cells (Grinvald et al. 1988; reviewed by Cohen 1973). To determine whether dye-independent signals contributed to voltage image waveforms, recordings were performed at 710 and 580 nm. In the action spectrum of the dye RH-155, the transmitted light intensity was expected to decrease (absorption increased) at 710 nm and increase (absorption decreased) at 580 nm. Indeed, similar to previous reports (e.g., Momose-Sato et al. 1999), changing the light wavelength from 710 to 580 nm reversed the signals and decreased their amplitude (n = 3, data not shown). Moreover, dorsal root–evoked optical signals were not evident in unstained slices (n = 4), suggesting that images recorded in the stained slice reflected voltage-related changes in dye absorption. Voltage images presented in our study were averages of three to five stimuli at 0.1–0.2 Hz. The signal (I/I) was of the order of 10–3.
Distinct optical waveforms were recorded along the dorsoventral axis of the slice. Typically, at stimulus intensities that evoked ventral horn responses, a spike-like signal followed by a large, slow component that decayed over several tens of milliseconds was recorded in the dorsal horn (Fig. 3; n = 52). Similar dual-component images were also apparent in the intermediate areas in approximately one-half the slices (n = 24/52), but these were smaller than signals recorded in the dorsal horn. In contrast, only small, slow optical responses with latencies of 4–7 ms were recorded in the ventral horn of all slices. Dual-component voltage images have been recorded in the spinal cord of chick embryos (Arai et al. 1999; Mochida et al. 2001), and based on pharmacological manipulations, it has been suggested that the spike-like component corresponded to presynaptic action potentials, and the slow response originated from synaptic potentials. To characterize the spatial distribution of dorsal root afferents, transverse slices were stained with antivesicular glutamate transporter1 (VGLUT1) antibody that is expressed in glutamatergic projections from cutaneous and muscle mechanoreceptors in the rat spinal cord (Alvarez et al. 2004). The spatial architecture of VGLUT1 immunoreactivity varied in different lumbar segments, but similar to the report by Alvarez et al., in all segments, the highest density was evident in the dorsal horn and the lateral intermediate area (n = 3, data not shown), where spike-like signals were frequently recorded.
Close correlation between dorsal root–evoked voltage images and subthreshold postsynaptic potentials
The interpretation that spike-like optical signals represented presynaptic action potentials and the slow signals were related to postsynaptic potentials remained speculative in the absence of direct measurements quantitatively relating voltage-sensitive dye signals to neuronal membrane potentials. To relate local voltage images to neuronal membrane potentials, simultaneous whole cell recordings were performed at targeted sites. The presence of the whole cell recording pipette did not distort the optical signals, because similar waveforms were measured in the presence and absence of the pipette. Similar resting membrane potentials (more negative than –50 mV) and action potential waveforms were recorded in stained and unstained slices, indicating that the dye did not significantly affect membrane properties. It is reasonable to assume that the dye did not change synaptic transmission, because the frequency of spontaneous synaptic potentials (0.4–1.7 Hz) was similar to that recorded in our previous studies (e.g., Gao et al. 1998).
Recordings were performed with a x20 objective, and because of its large dimensions, measurements were restricted to sites 
100 µm away from the stimulating electrode. Therefore in the majority of the experiments, recordings did not include neurons in laminae I and II. Most recordings in the ventral horn were performed in lamina VII interneurons and did not include neurons in laminae VIII and IX (motor nuclei) because of the noisy optical signals near the edge of the slice (Fig. 2). Optical signals primarily originated from neurons in focus (superficial), but presumably potential changes in neurons in deeper layers also contributed to the signals. The x20 objective had a 30-µm-diam optical field, and with a cell diameter of 20–25 µm, it was estimated that in the 500-µm-thick slice, each diode received light from 20 cell bodies and an unknown number of dendrites and axons.
Dorsal root stimulation at intensities that generated optical responses in the ventral horn produced postsynaptic potentials in neurons located in most regions of the cord (n = 24). Although voltage images around the soma presumably resulted from potential changes in a set of neurons, the time-course of optical responses with duration >100 ms (from peak response to its return to baseline) was closely correlated with the time course of subthreshold synaptic potentials (Fig. 4; n = 18).
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; Ziskind-Conhaim 1990). Modulation of dorsal root–evoked optical responses by excitatory and inhibitory synapses
To determine the contributions of excitatory and inhibitory synaptic transmission to voltage image waveforms, synapses were blocked with specific receptor antagonists. Blocking glutamate receptors by D-APV (20 µM) and CNQX (10 µM), NMDA and non-NMDA receptor antagonists, significantly reduced the amplitude of the large, slow component in the dorsal horn and intermediate area and completely blocked the small signal in the ventral horn (Fig. 8; n = 9). Blocking glutamatergic synaptic transmission inhibited dorsal root–evoked EPSPs in all regions of the cord (n = 5, data not shown). These findings suggested that a major component of the slow optical signals was generated by glutamate-mediated EPSPs. Glutamate receptor antagonists did not affect the amplitude of the spike-like component in the dorsal horn but reduced it in two of five recordings in the intermediate area (Fig. 8). It is conceivable that the fraction of D-APV–and CNQX-sensitive spike-like component in the intermediate region corresponded to postsynaptic action potentials.
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The contribution of inhibitory synapses to the properties of dorsal root–evoked voltage images and whole cell potentials was examined by blocking glycinergic and GABAergic synaptic transmission using specific concentrations of glycine and GABAA receptor antagonists (Gao et al. 2001; Hinckley et al. 2005b). Exposure to strychnine (0.5 µM) induced an average increase of 47% in the amplitude of the optical responses (Fig. 9A; n = 11). The increase in optical signals was closely correlated with an increase in EPSP amplitude (n = 4). In one neuron, the larger EPSP reached threshold for action potential (Fig. 9A), but as shown in Fig. 5A, the action potential was not correlated with a spike-like optical signal.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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Optical signals from voltage-sensitive dye in the rat spinal cord
Voltage images of spontaneous and evoked activity in the embryonic chick spinal cord have provided valuable information regarding the ontogeny of neuronal excitation with relation to functional network organization and properties of newly formed neural networks (Arai et al. 1999; Ikeda et al. 1998; Mochida et al. 2001). Only recently fast, voltage-sensitive dyes have been employed to monitor electrical activity in the postnatal rodent spinal cord. A complex spatiotemporal pattern of spontaneous oscillations was apparent in transverse sections of the neonatal rat spinal cord with depolarizations alternating between mirror locations in the right and left sides of the cord (Demir et al. 2002). Voltage images have also been used in an attempt to characterize the patterns of network activity during neurochemically induced locomotor-like rhythms in the neonatal mouse spinal cord (Arai et al. 2002, 2003). The spatiotemporal pattern of induced rhythms could not be clearly resolved in the cord except for the motor nuclei, in which rhythmic optical signals were closely correlated with ventral root alternating rhythms. It is reasonable to assume that rhythmic voltage images in the motor nuclei are reflective of the synchronous excitation of clustered motoneurons. The inability to detect rhythmic optical signals in other regions of the cord raises the intriguing possibility that interneurons constituting part of the rhythm-generating networks do not aggregate in large clusters (Hinckley et al. 2005a).
Functional interpretation of optical signals can be complicated in the absence of direct comparison with transmembrane potentials, because voltage images consist of extrinsic, dye-dependent signals and intrinsic, dye-independent signals. Under our experimental conditions, as in the neonatal mouse spinal cord (Arai et al. 2002), dorsal root–evoked images were dye-dependent, reflecting voltage-related changes in dye absorption. Interestingly, Arai et al. have shown that, in the same preparation, induced locomotor-like optical signals recorded with voltage-sensitive absorption dye are dye-independent and therefore are not related to changes in transmembrane potentials. Close correspondence has been shown between optical and electrical responses to dorsal and ventral root stimuli in chick spinal motoneurons antidromically labeled with voltage-sensitive dye (Wenner et al. 1996). Similarly, we have shown that the spontaneous optical oscillations that alternate between the left/right sides of the rat spinal are closely correlated with extracellular recordings (Demir et al. 2002).
Properties of dorsal root–evoked voltage images
Dorsal root–evoked optical responses consisting of dual-component fast and slow voltage images and single-component slow signals were similar to those recorded in the chick spinal cord. The latencies of the slow voltage images in the intermediate area and the ventral horn varied from 4 to 7 ms. Although it is likely that these slow images corresponded to polysynaptic EPSPs, we cannot rule out the possibility that some were correlated with monosynaptic EPSPs. Nerve- and dorsal root–evoked monosynaptic EPSPs with latencies ranging from 3 to 5 ms were recorded in spinal motoneurons of the neonatal rat (Seebach and Ziskind-Conhaim 1994; Ziskind-Conhaim 1990). It has been suggested that the spike-like signal was reflective of action potentials in afferent axons, whereas the slow optical signal was related to postsynaptic potentials (Arai et al. 1999; Mochida et al. 2001; reviewed by Momose-Sato et al. 2001). Our finding supports this assumption, because the spike-like optical component was not correlated with postsynaptic action potential. Based on our preliminary immunohistochemical observations that the highest density of afferent projections was in the dorsal horn and the lateral intermediate area, it is reasonable to assume that spike-like signals in those areas originated from synchronous firing in dorsal root afferents.
The time-course of slow optical signals was remarkably similar to that of whole cell subthreshold membrane potentials. This is somewhat surprising because voltage images originated from membrane potential changes in numerous cell bodies, dendrites, and axons in the area around the soma. Strong correlation between sensory-evoked optical signals and subthreshold synaptic potentials in single neurons has also been shown in layer 2/3 pyramidal neurons in the rat somatosensory barrel cortex in vitro and in vivo (Petersen and Sakmann 2001; Petersen et al. 2003). These studies indicated that the high temporal resolution of voltage-sensitive dye imaging can reflect with a single-cell time-course the synchronous subthreshold excitation of group of neurons in the optical field.
Blocking glutamatergic synaptic transmission suppressed both synaptic potentials and the slow images in the ventral horn, indicating that those were glutamate-mediated potentials. However, inhibiting glutamatergic synapses only partially blocked the large, slow optical response that followed the spike-like component in the dorsal horn and intermediate area. We made no attempt to characterize the D-APV–and CNQX-resistant component. It is conceivable that this signal corresponded to a slow Ca-dependent afterdepolarizing potential similar to that described in motoneurons in the postnatal rat spinal cord (Walton and Fulton 1986). An alternative possibility is that the component is reflective of glial depolarization (Kojima et al. 1999; Konnerth and Orkand 1986; Lev-Ram and Grinvald 1986). Optical signals from parallel fibers in the cerebellum of the skate consisted of a spike-like component and a larger, slow component that lasted >400 ms (Konnerth et al. 1987). The slow component that was recorded when using the voltage-sensitive absorption dye RH-155 but not RH-482 was attributed to accumulation of extracellular potassium and glial membrane depolarization. We cannot rule out the possibility that the slow component recorded in our experiments originated from glial depolarization. However, the close correspondence of changes in amplitude and time-course of EPSPs with changes in optical signals suggest that a significant fraction of that signal originated from subthreshold excitatory postsynaptic potentials.
GABAergic and glycinergic synapses regulated in a similar manner the amplitude and time-course of both EPSPs and voltage images. Disinhibition increased the amplitude of both optical signals and EPSPs and blocking GABAergic synaptic transmission also prolonged their duration. The prolonged duration might be attributed to the slow decay time constant of GABAA receptor–mediated synaptic currents (Gao et al. 2001; Jonas et al. 1998). Similar to the distinct actions of glycinergic and GABAergic inhibition on dorsal root–evoked voltage images and EPSPs, we have recently shown that exposure to glycine and GABAA receptor antagonists affect differently neurochemically induced locomotor-like rhythms in the mouse spinal cord (Hinckley et al. 2005b). Blocking GABAA receptor–mediated synapses synchronized the onset and prolonged the duration of rhythmic discharges, whereas blocking glycine receptors increased tonic discharges and reduced the phase correlation between the alternating rhythms.
Our study showed that dorsal root–evoked local voltage images are closely correlated with EPSPs, indicating that the imaging technique can be effectively used to monitor subthreshold membrane potentials in targeted areas in the postnatal rodent spinal cord. Moreover, the temporal resolution of optical signals from voltage-sensitive dyes provides valuable information regarding the sites of synchronous firing in presynaptic neurons.
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. Ziskind-Conhaim, Dept. of Physiology, 129 SMI, Univ. of Wisconsin, Medical School, Madison, WI 53706 (E-mail: lconhaim{at}physiology.wisc.edu)
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REFERENCES |
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