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1The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami 33101; 2Department of Biomedical Engineering, University of Miami, Miami, Florida 33124; and 3Department of Biomedical Sciences, University of Illinois College of Medicine, Rockford, Illinois 61107-1897
Submitted 27 October 2003; accepted in final form 2 March 2004
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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1 h to estimate contributions to resting levels from tonic descending activity and to delineate chemical changes that may influence the degree of pathology and recovery after spinal injury. Measurements employed fast cyclic voltammetry with carbon fiber microelectrodes to give high spatial resolution. Monoamine oxidation currents, sampled at equal vertical spacings within each segment, were displayed as contours over the boundaries delineated by histologically reconstructed electrode tracks. Monoamine oxidation currents were found in well defined foci, often confined within a single lamina. Larger currents were typically found in the dorsal or ventral horns and in the lateral aspect of the intermediate zone. Cooling of the low-thoracic spinal cord led to a decrease in the oxidation current (to 71–85% of control) in dorsal and ventral horns. Subsequent low-thoracic transection produced a transient increase in signal in some animals followed by a longer lasting decrease to levels similar to or below that with cooling (to 17–86% of control values). We conclude that descending fibers tonically release high amounts of monoamines in localized regions of the dorsal and ventral horn of the lumbar spinal cord at rest. Lower amounts of monoamines were detected in medial intermediate zone areas, where strong release may be needed for descending activation of locomotor rhythms. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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; Barbeau and Rossignol 1991; Kiehn et al. 1992; Marcoux and Rossignol 2000) and of modulating nociceptive transmission (Millan 1999, 2002; Willis and Westlund 1997). Because virtually all spinal monoaminergic innervation originates supraspinally (Basbaum et al. 1978; Carlsson et al. 1963; Clarke and Proudfit 1991a,b; Grzanna and Fritschy 1991; Martin et al. 1978; Westlund et al. 1982), spinal cord injury inevitably affects the normal function of these spinal systems. However, spinal neurons retain functional postsynaptic receptors on their cell membranes following denervation (Giroux et al. 1999; Noga et al. 2002), so that monoamine transmitter replacement therapies may potentially improve locomotor function (Feraboli-Lohnherr et al. 1997; Yakovleff 1995) and alleviate chronic pain (Hentall and Sagen 2000; Horiuchi et al. 2003) following spinal cord injury.
We have recently measured monoamine release in the spinal cord during evoked locomotion (Noga et al. 1999) and stimulation of monoaminergic centers in the brain stem (Hentall et al. 2003a,b). These preliminary findings gave the first indications of the temporal and spatial patterns of changes in monoamine concentration in the spinal cord following activation of neurons of the noradrenergic dorsal pons and the serotonergic medullary raphe. Such data are essential both for delineating basic mechanisms of spinal monoaminergic transmission and for determining potential targets of monoamine transmitter replacement therapies. Our findings confirmed that the actions of monoamines in the spinal cord likely occur through nonsynaptic diffusion as well as classical synaptic neurotransmission (Bach-y-Rita 1999; Marlier et al. 1991; Maxwell et al. 1983; Ridet et al. 1994; Vizi 2000).
Here we examine the distribution of monoamines released from terminals of descending monoaminergic fibers in the rat lumbar spinal cord in the absence of behavioral activation (i.e., at "rest"). In this study, a spatial mapping technique (Noga et al. 1995) was combined with fast cyclic voltammetry (FCV) (Armstrong-James and Millar 1984). The in vivo voltammetry technique is based on the ability of some substances such as monoamine neurotransmitters and their metabolites to oxidize at the surface of carbon fiber microelectrodes (CFME) when an appropriate potential is applied to the electrode. This results in a release of electrons at the electrode surface that may be measured as a current, the amount being proportional to the number of molecules oxidized. In contrast to extractive techniques of measurement such as microdialysis combined with HPLC, fast voltammetry with CFMEs allows one to determine extracellular neurotransmitter content with very high spatial and temporal resolution. However, since voltammetric measurements must be referred to some initial starting value, it is easier to map changes, such as those due to brain stem stimulation, than to map basal quantities. Here we solve this difficulty by using dorsal white matter as a spatial reference point, where relative concentrations are likely to be much lower due to the absence of nearby release sites. The work achieved three main goals. First, it indicated the basal monoamine levels on which dynamic descending effects are superimposed. Second, it showed the extent to which tonic descending activity determines steady-state monoamine levels. Third, it revealed how spinal monoamine levels change during the first hour after spinalization, which has important implications for neuroprotection (Salzman et al. 1994), neuronal regeneration (von Euler et al. 2002), pain (Horiuchi et al. 2003), and hyperreflexia (Bennett et al. 2001) following spinal cord injury. A key experimental procedure for the last two aims was voltammetric measurement from selected areas within the lumbar spinal gray matter during and after cooling and transection of the thoracic spinal cord. Preliminary results have been presented previously (Noga et al. 2001).
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METHODS |
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Experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publications No. 80-23; revised 1996). The number of animals used, and their pain and distress, was minimized.
Adult male Fisher rats (n = 9, Fisher-CDF strain, Charles River Labs.), weighing 250-300 g, were anesthetized with 1–3% halothane in a mixture of 60% nitrous oxide and 40% oxygen. Animals were first placed in a closed chamber for anesthetic induction, and subsequently, the halothane mixture was applied through a facemask. The trachea was intubated for direct anesthetic administration. The left common carotid artery was cannulated and connected to a pressure transducer for blood pressure monitoring. The right external jugular vein was cannulated for the administration of fluids (normal saline solution for fluid loss and bicarbonate solution with 5% glucose to maintain a normal pH balance). Body temperature was maintained at 37°C using feedback-controlled heating lamps. CO2, O2, and tissue oxygenation were monitored throughout the experiment using a Datex OscarOxy airway gas monitor. The levels of anesthetic were adjusted to ensure that there was no cardiac or respiratory depression. Withdrawal reflexes were also monitored to ensure that the animal was appropriately anesthetized. A multi-level laminectomy was performed to expose the lumbar spinal cord. In five animals, the sixth and seventh thoracic segments were also exposed for acute transection at a later time during the experiment. The animals were transferred to a stereotaxic headframe and spinal cord fixation device. All four limbs were pendant. A back pool was formed with the skin flaps, and the muscle surrounding the spinal cord area was covered with Reprosil (Dentsply Caulk, Milford, DE). The spinal cord was covered with warmed saline, and the temperature of the pool was maintained at 37°C using a feedback-controlled heating lamp. Voltammetric measurements were made in the L1–L5 segments (see In vivo voltammetry and isocurrent mapping). In the five spinalized animals, the T6/7 cord segments were exposed by cutting and retracting the dura, and in three cases, were cooled with crushed ice prior to transection. The cord was transected in the medial-to-lateral direction using spring-mounted scissors under microscopic guidance. The completeness of the transection was confirmed visually by the slight separation of the proximal and distal stumps of the cord. Gelfoam was applied between the transected stumps when necessary to stop bleeding. No displacement of the cord was observed distally at the level of the recording site when the electrode was withdrawn at the end of the experiment.
FCV and CFMEs
CFMEs based on a 33-µm-diam glassy carbon monofilament were constructed to established procedures (Armstrong-James and Millar 1984; O'Connor and Kruk 1991) and electrically pretreated as described previously (Hentall et al. 2003a). A Millar Voltammeter (P.D. Systems International) was used to generate the FCV scans. The Millar Voltammeter uses a three-electrode potentiostat design including a CFME, a 5-cm-long Ag-AgCl auxiliary electrode, and a carbon-based reference electrode, internally filled with KCl (Dri-Ref, WPI, Sarasota, FL). The oxidation of monoamines was caused by applying voltage ramp waveforms lasting 20 ms (from 0 to –1 to +1.4 to –1 to 0 V) at a scan rate of 450 V/s, repeated at a frequency of 1 Hz. The output voltage (or "voltammogram") signaled the clamping current in the CFME. This was in the form of a "subtracted voltammogram," obtained by digitally (at 10–20 kHz) subtracting a reference voltammogram recorded at the cord surface and stored as a background signal.
The electrodes were calibrated ex vivo (Fig. 1) in 0.25–2.5 µM of 5-HT, DA, and NE. The following monoamine metabolites and other potential contaminants were also tested in 25- to 250-µM concentrations: 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA), 3,4-dihydroxyphenylacetic acid (DOPAC), 4-hydroxy-3-methoxy-phenyl(ethylene)glycol (MHPG), and the purine derivative, uric acid (UA). All substances were dissolved in phosphate-buffered saline (PBS) at pH 7.4. Calibrations were always done after each experiment since insertion into brain tissue significantly decreases electrode sensitivity (Stamford et al. 1992). In the presence of monoamines, oxidation currents were generated during the first ascending phase of the triangular input waveform. Oxidation peaks of all tested monoamines, which were maximal when the applied input waveform was around 950 mV, could not be distinguished from each other (Fig. 1). The sensitivity of the electrodes (n = 11) in nM/nA was 12.7 ± 2.5 (SE) for 5-HT, 47.9 ± 9.0 for DA, and 118.7 ± 17.4 for NE. The CFMEs were much less sensitive to anionic metabolites of the transmitters (Commissiong 1985a,b; Kopin 1985; O'Neill et al. 1998). Specific values were as follows: 5-HIAA, 830 ± 105 (n = 7); HVA, 919 ± 216 (n = 4); DOPAC, 5073 ± 888 (n = 7); and MHPG, 692 ± 19 (n = 3) nM/nA. The sensitivity to UA was also low: 2225 ± 407 (n = 2). Postoxidation reduction currents of catecholamines and indoleamines showed quite different patterns, which was used as an aid to their identification. Catecholamines had single peaks near –400 mV, whereas indoleamines showed two peaks: one near –100 mV and the other near –800 mV (Fig. 1). Reduction peaks were always of smaller amplitude, probably because diffusion of the products prevented complete reversal of the electrode oxidation reaction.
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). A physiologically large decrease of 0.1 units in the presence of NE enhanced the current output across most of the voltage scan. Ascorbate slightly enhanced the oxidation current but did not alter the postoxidation reduction current. In vivo voltammetry and isocurrent mapping
FCV measurements were made at various recording sites within the L1–L5 spinal segments of halothane anesthetized rats (Fig. 2) following localized removal of the pia matter. The reference electrode was positioned near the recording site in the spinal cord, and the auxiliary electrode was covered with saline-soaked gauze and placed into a nearby muscle. Changes in the concentration of the various monoamines at the different recording locations within the spinal cord were measured relative to the initial surface scan of each track (Fig. 2B, top). The depth of the electrode in all tracks in a given mapping session was referred to the surface depth of the first track. This was done so that measurements at each depth were always related to ones obtained at similar depths in the adjacent tracks. In penetrations where the surface of the spinal cord was different from the initial track, the first subtracted voltammogram was obtained slightly above the spinal cord (Fig. 5). The electrodes were advanced in steps of 2 µm at 1 Hz along vertical penetrations through the spinal cord (Fig. 3A). Signal amplitudes of oxidation peaks were stable at each depth when electrodes were advanced at this slow rate. In some cases, voltammetric scans recorded from each point were averaged in 100-µm segments and assigned to the average depth. In other experiments, the microelectrode was stopped every 100 µm (Fig. 3B), and 20 triggered sweeps were averaged. Tracks were equally spaced in the lateral direction (every 100 µm) and were made successively from the most medial point to the most lateral point possible on the dorsal surface of the spinal cord. The final depth of penetration was selected to prevent microelectrode damage.
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) and color-coded maps of the oxidation current generated. The histologically reconstructed maps of recording sites were re-scaled for tissue shrinkage and the angle of microelectrode penetration and superimposed on the cross-sectional isocurrent contour maps. In cases where subtracted voltammograms obtained every 2 µm were averaged together for every 100 µm of the matrix (Fig. 3A, track 4), the midpoint depth was used when overlaying the histological outline onto the coordinates of the matrix, and the maps appear to start slightly below the surface of the cord (Fig. 5, E, F, H, and K).
In the experiments with cooling and transection of the thoracic spinal cord, CFMEs were positioned at locations within the dorsal or ventral horns where oxidation peaks were large. Voltammograms applied every second were captured before, during, and after cooling and/or spinalization. A xanthine oxidase inhibitor allopurinol (10 mg/kg dissolved in 1.0 ml of 0.9% NaCl) was slowly administered (over 100 s) intraarterially (ia) to one animal to diminish the concentration of UA, a potential contaminant of the oxidation signal (Rivot et al. 1987). Allopurinol was only weakly electroactive (4065 nM/nA).
Histology
At the end of the experimental procedure, the cords were removed and immersed in 4% paraformaldehyde in PBS solution (pH 7.4). Frozen serial sections (50 µm) of the relevant spinal cord segments were cut on a sliding microtome, collected in PBS, and counterstained with toluidine blue or cresyl violet (Hentall et al. 2003a). Camera lucida drawings were made to reconstruct the position of the electrode tracks. The distance between histologically observed electrode tracks was used to calculate shrinkage.
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RESULTS |
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Peaks occurring in voltammograms during the first positive going ramp of the applied triangular waveform were delayed in vivo from those acquired ex vivo (Fig. 2) (Hentall et al. 2003a), occurring at 1035 ± 7.1 (SE) mV. The peaks were also somewhat broader than those seen ex vivo. They were nevertheless taken to represent a monoamine signal because similar signals were produced by microinjection or superfusion of monoamines while recording within the dorsal horn (see Fig. 2A for response to 5-HT). Reduction currents were not always clearly observed and were delayed with respect to ex vivo calibrations. Reduction peaks of topically applied or intraspinally injected monoamines, when present, were also delayed. It was usually not possible to distinguish between indoleamines or catecholamines from reduction currents in vivo. The postoxidation reduction current recorded in vivo typically showed a single peak (Fig. 2). This could be due to a mixture of indoleamines and catecholamines since the single reduction peak of catecholamines is found midway between the double reduction peaks of indoleamines (Fig. 1A).
Basal mapping
Peaks in the amplitude of the oxidation current were observed at various depths within the gray matter during electrode tracking. The localized nature of the peaks observed as electrodes were advanced downward were confirmed with measurements made at equal increments while the electrodes were withdrawn upward at the same speed from the spinal cord (n = 10). As can be seen from a representative example in Fig. 4, local maxima in monoamine oxidation current within the dorsal horn (at depths of 400, 850, and 1130 µm), observed when the CFME was inserted, were also observed when the electrode was withdrawn. The amplitude of the oxidation current, however, was decreased during withdrawal.
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With regard to segmental differences, the largest oxidation peaks in L1 were observed predominantly in lateral areas of laminae II, III, and V. Smaller foci were found in the lateral or central portion of laminae IV, V, and VII–VIII (Fig. 5, A and B). In L2, the largest oxidation currents were observed in medial or central portions of laminae I–III, VIII, and IX. Lesser currents were found in central-to-lateral parts of laminae IV, V, VII, and lateral lamina IX (Fig. 5, C and D). In L3, a zone of high oxidation current was observed along the medial aspect of the entire dorsal horn (Fig. 5E) and in central-to-lateral portions of laminae IV–IX (Fig. 5, F and G). Zones of lesser current were found separating these areas (Fig. 5, E–G). In L4, the largest foci were located primarily along medial areas of the spinal gray matter in laminae I–IV and in the medial nucleus of lamina IX, with lower currents in the central and lateral parts of laminae IV–VII (Fig. 5, H–J). In L5, areas of highest oxidation currents were found along lateral aspects of the dorsal horn, intermediate zone, and ventral horn and in laminae IV/V (medial), VIII, and the medial nucleus of lamina IX (Fig. 5, K and L).
Interruption of descending activity
The contribution of neurotransmitters released from tonically active descending monoaminergic pathways to oxidation currents measured either within the dorsal or ventral horns was determined by cooling and/or transection of the thoracic spinal cord. The results are shown in Fig. 6. Cooling of the spinal cord with crushed ice (n = 3) decreased the oxidation signal recorded within the dorsal horn to 71–85% of control values (Fig. 6, A, C, and D). Transection of the cord usually resulted in a brief (20–45 s) injury-evoked increase in the oxidation signal (Fig. 6, A, B, D, and E), followed by a drop in the oxidation current amplitude to levels lower than pretransection values, either immediately (Fig. 6, C–E) or after a short delay (Fig. 6, A and B). Oxidation current amplitudes attained a low of 17–86% of control values (mean 57.4 ± 11.9%, n = 5). In one case, oxidation current values returned to normal control (precooling) values, 72 min after transection (Fig. 6A). The maximal current drop was 64, 125, 47, 29, and 10 nA for Fig. 6, A–E, respectively.
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1,200, 1,200, 500, 200, and 200 nM, respectively, as determined from ex vivo calibrations. The highest concentrations of monoamines released from tonically active descending fibers were thus in laminae I–III, with progressively lower amounts released in more ventral areas of the dorsal horn (medial lamina IV) and in the intermediate zone (laminae VII). Following transection, residual currents of 138, 25, 40, 69, and 59 nA were observed for Fig. 6, A–E, respectively. An upper limit on the concentration of metabolite at each of these sites can be obtained if one assumes that residual currents originate from the monoamine metabolites (see DISCUSSION below). Assuming that monoaminergic transmitter metabolites are present in the same ratio as their parent transmitters, that the CFMEs had similar sensitivities to 5-HIAA, MHPG, and HVA in these five experiments (700 nM/nA), and that contributions from DOPAC can be excluded by virtue of its very low oxidation current ex vivo, the concentrations for 5-HIAA and MHPG at these sites was estimated to be 39, 7, 11, 19, and 17 µM (with one-half that amount for HVA), respectively. Allopurinol administration (Fig. 6D) resulted in a brief rise in measured current, likely due to the weak oxidation current that this substance generates by itself. Subsequently, an additional 5-nA (5%) drop in oxidation current (11.1 µM) was noted. |
DISCUSSION |
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) with FCV to measure extracellular concentrations of monoamines within the lumbar spinal cord of the anesthetized rat. Salient findings included large gradients in oxidation current, which can go from highest to lowest in 100–200 µm. Such high gradients were not detected in previous studies, probably because measurements were made either with microdialysis probes (e.g., Bowker and Abhold 1990; Lisi et al. 2003; Matos et al. 1992; Men et al. 1996; Sorkin et al. 1991) or larger carbon fiber electrodes (Rivot et al. 1983, 1995) that span several spinal laminae. Also, a significant amount of the monoamine content was shown to be released from tonically active descending pathways. However, transection of the spinal cord led initially to a transient increase in monoamine levels, which may be a significant factor in the induction of acute and chronic neurological response to spinal cord injury. What substances contribute to the steady-state oxidation currents within the lumbar cord?
These voltammetric data show high spatial resolution, but the chemical species contributing to the signal are best deduced in conjunction with previous analyses using chemical extraction (principally microdialysis) and with the known chemical neuroanatomy. Measurements of endogenous monoamine levels in the rat, using tissue extracts (Basbaum et al. 1987; Commissiong 1985b; Commissiong et al. 1979, 1984; Ko et al. 1997) or in vivo microdialysis (Abhold and Bowker 1990; Bowker and Abhold 1990; Lisi et al. 2003; Men et al. 1996), indicate that the spinal 5-HT concentration is several times higher than DA and is relatively similar to NE. Our electrodes were more sensitive to 5-HT than to NE or DA (9 and 4 times more, respectively). Thus 5-HT is likely to have produced most of the current in the electrochemical signal. In support of this view, the largest oxidation currents were also observed where descending serotonergic terminals are most dense (Jones and Light 1992; Maxwell et al. 1983, 1996).
NE probably also provided a significant proportion of the oxidation current at many sites, even if less than that provided by 5-HT. The spinal cord is densely innervated by descending noradrenergic fibers in the dorsal horn (laminae I–IV), intermediate zone, and ventral horn (lamina IX) (Clarke and Proudfit 1991a,b; Grzanna and Fritschy 1991), which are all regions where large oxidation currents were observed. DA, on the other hand, probably contributed less to the electrochemical signal given the relatively low levels reported previously, although we cannot rule out that a few individual oxidation foci were due to DA release. Dopaminergic pathways descend within the dorsolateral funiculus (Commissiong et al. 1979; Skagerberg et al. 1982) and terminate in the dorsal horn (primarily laminae III–V), the intermediolateral cell column, close to the central canal, and sparingly within the ventral horn (Ridet et al. 1992; Skagerberg et al. 1982).
A significant portion of the total current could also have come from the 5-HT metabolite 5-HIAA, even though the electrodes were 65 times less sensitive to 5-HIAA than to 5-HT. 5-HIAA is found in much higher concentrations than 5-HT in dialysates from the dorsal horn (Matos et al. 1992; Men and Matsui 1994; O'Neill et al. 1998; Sorkin et al. 1991). If the amounts of serotonergic and noradrenergic metabolites were in equal concentrations (Commissiong et al. 1984), and only metabolites were present following transection, posttransection measurements suggest that levels of 5-HIAA and MHPG (the major metabolite of NE) were between 7 and 39 µM. For 5-HIAA, this is in general agreement with differential pulse voltammetry measurements of indoleamines in the rat dorsal horn (Rivot et al. 1987, 1995). The assumption that only metabolites remain after transection is based on the improbability of continued transmitter release from the terminals of the severed monoaminergic projections due to injury-evoked depolarization, since severed axons seal within 30–60 min (Shi et al. 2000). The steady drop in oxidation current over the 1-h period following transection could reflect this sealing. It is possible, however, that monoamine uptake mechanisms are reduced after transection due to energy deprivation. No evidence is available on this point, and nonmyelinated axons are less vulnerable than larger axons to oxygen and energy deprivation following injury (Shi and Pryor 2002). Another untested possibility is an extraneuronal (vascular) source of monoamines (Salzman et al. 1987) that would maintain transmitter levels within the distal cord immediately after injury.
Metabolites of DA are found in much higher concentrations in the rat dorsal horn compared with DA (Men and Matsui 1994), but our CFMEs were 19 and 106 times less sensitive to HVA and DOPAC than to DA, and extracellular dopaminergic metabolites are at much lower levels than 5-HIAA (Matos et al. 1992; Men and Matsui 1994). NE and DA, being cations, diffuse slower than their anion metabolites (Rice et al. 1985), supporting the notion that the present highly differentiated maps mostly reflect the primary neurotransmitter.
Other neurotransmitters, principally amino acids or peptides, do not contribute to the current registered by FCV (Stamford et al. 1992). UA, a product of purine metabolism found within the spinal cord (Basbaum et al. 1987; Rivot et al. 1987, 1995), and ascorbate are more likely to be contaminants. Our CFMEs were weakly sensitive to UA (185 times less than to 5-HT). Also, allopurinol administration resulted in only a 5% drop in the oxidation current within the dorsal horn (Fig. 6B). Hence the contribution of UA to the recorded signals within the spinal cord is likely to be low. High concentrations of ascorbate enhance the oxidation current generated by monoamines (Hentall et al. 2003a). However, powerful homeostatic mechanisms maintain ascorbate at relatively fixed levels in the extracellular space (Schenk et al. 1982), so this substance is unlikely to have given rise to the present spatially differentiated map.
Functional considerations
Oxidation current foci were found in all laminae, with little intersegmental differences in the regions examined. Thus several descending functions appear to be continuously operative. One is the tonic control of vasomotor tone and arterial pressure (Millan 2002) within the intermediolateral horn of the thoracic and upper lumbar spinal cord, where terminals of monoaminergic pathways innervating preganglionic sympathetic neurons are found (Fuxe et al. 1990; Hosoya et al. 1991). In laminae I, II (outer), IV, V, and VI, the high concentrations of monoamines may be related to their importance in the control of nociceptor transmission from C and A
fibers that project to these areas (Craig and Dostrovsky 1999; Millan 1999) and in lamina IX to the control of motoneuron activity (Schmidt and Jordan 2000). The medial part of lamina VII, on the other hand, showed low oxidation currents, despite the concentration of monoaminergic terminals here. This can be explained by the fact that neurons in this area of the rat (Kjaerulff and Kiehn 1996), like the cat (Huang et al. 2000; Johnson et al. 2002; Noga et al. 1995), are involved in the control of walking and other voluntary movements, functions that are only intermittently required.
The highest monoamine concentrations observed (in the superficial dorsal horn) were well above those needed by endogenous ligands to activate serotonergic and catecholaminergic receptors (Bunin and Wightman 1998; Callado and Stamford 2000; Cragg et al. 2001). Given that the CFME cannot resolve concentrations confined to the synaptic cleft, this implies that nonsynaptic "volume" or diffusion neurotransmission is likely (Bach-y-Rita 1999; Vizi 2000). This concept is supported by observations that serotonergic and noradrenergic terminals within the dorsal horn of the rat exhibit little synaptic specialization (Marlier et al. 1991; Maxwell et al. 1983; Ridet et al. 1993, 1994). In the ventral horn, in contrast, synaptic contacts are predominant for serotonergic terminals (Privat et al. 1988), so that serotonergic volume transmission may here be less important. The well-defined oxidation current maxima, however, and the steep concentration gradients indicate that transporter uptake likely restricts neurotransmitter diffusion a few hundred microns at most.
Effects of acute spinal transection
Spinal cooling or transection resulted in a complex pattern of rise and fall in the oxidation currents within the spinal cord. The drops strongly suggest ongoing release from terminals of descending monoamine pathways, as shown by microdialysis studies (Abhold and Bowker 1990; Bowker and Abhold 1990; Lisi et al. 2003; Men et al. 1996). The reduction amounted on average to 43% of the oxidation current (range, 14–83% of control values). Tonic activity through descending monoaminergic pathways is known to modulate segmental afferent activation of spinal neurons (Millan 1999, 2002; Rivot et al. 1987). Cells within the monoaminergic nuclei frequently display a steady discharge rate (Faiers and Mogenson 1976; Fields et al. 1983; Valentino and Aston-Jones 1995) that could account for the release of these transmitters at the spinal level. Surgical exposure of the spinal cord should result in increased noxious stimulation of multireceptive dorsal horn neurons (Li et al. 1998) and increased activation of descending monoaminergic pathways (Dickenson and Goldsmith 1986; Valentino and Aston-Jones 1995), with consequent increased monoamine release. This idea is supported by the observation that peripheral nerve stimulation can increase the release of 5-HT, NE, and DA within the spinal cord (Men and Matsui 1994; Men et al. 1996). The well-defined foci of monoamine oxidation current observed here may therefore reflect recruitment of descending monoaminergic pathways causing "localized alterations in nociceptive processing" (Millan 2002). Such foci may be further enhanced by the anesthetic (Keita et al. 1999; Pashkov and Hemmings 2002).
The transient changes in the extracellular monoamine signal in the distal stump of the cord over the first few hours following spinal cord injury have not previously been reported. However, the increases observed are not entirely new, since a small increase of 5-HT or related compounds (but not NE or DA) has been observed near cord impact injuries (Liu et al. 1990; Salzman et al. 1987). The rapid and massive efflux of monoamines seen here immediately after injury in distal cord segments is likely to affect both local vasomotor control (Saruhashi et al. 1990), and via its action on autonomic preganglionic neurons, arterial pressure (Fuxe et al. 1990; Hosoya et al. 1991; Millan 2002). 5-HT may also support the initiation of trophic effects that can influence fiber regeneration (Bregman et al. 2002; Salzman et al. 1994). On a longer time-scale, tissue levels of 5-HT and DA have been reported to rise 1 day following transection, then decrease to near zero levels by 5 and 9 days, respectively (Magnusson 1973). NE has been reported to show no initial increase but rather to decrease gradually to near zero levels by 15 days (Commissiong and Toffano 1989; Magnusson 1973).
In conclusion, high-resolution spatial mapping of monoaminergic substances with FCV provides insight into the dynamic nature of monoaminergic control of spinal function. The detection of spatial maxima and minima over distances as short as a few hundred microns indicate that monoaminergic control of sensory and motor function may be more precise than suggested by measurement techniques using larger diameter microdialysis probes or CFMEs with exposed lengths of several hundreds microns (which are essential for slow scanning across a range of oxidation potentials with pulse voltammetry). This implies that combining the spatial mapping of transmitter release with temporal measurements from multiple implanted CFMEs is a useful way to study the dynamic control of neural function by descending modulatory pathways in different states of behavioral activation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. R. Noga, The Miami Project to Cure Paralysis, Univ. of Miami School of Medicine, PO Box 016960, R-48, Miami, FL 33101 (E-mail: bnoga{at}miami.edu).
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