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Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
Submitted 4 October 2007; accepted in final form 8 May 2008
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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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and C fiber afferents and is transmitted to the thalamus, periaquaductal gray, and parabrachial area via lamina I ascending tract neurons (Craig 2000
). GABAergic neurons constitute 
25% of the neuronal population of lamina I (Polgar et al. 2003; Todd and Spike 1993). GABAergic neurons in this region are at a nodal point in the control of pain processing and have been shown to play an important role in spinal cord function and dysfunction, particularly in relation to pain (Coull et al. 2003) and spinal cord injury (Craig 2002; Finnerup and Jensen 2004). Because descending modulatory inputs to the dorsal horn are predominantly inhibitory (Gebhart 2004; Millan 2002), their loss after spinal cord transection likely contributes to hyperexcitability in spinal circuits that can manifest in increased nociceptive reflexes (Suzuki et al. 2004). Resulting hyperexcitability of dorsal horn neurons causes chronic pain (Malan et al. 2002; Millan 1999; Zeilhofer 2005). GABAergic neurons in lamina I are in a key position for the control of pain transmission. However, the ways in which these cells are affected by chronic spinal cord injury is unknown.
Voltage-gated ion channels, including Nav1.3, L-type Ca2+ (Cav1), and T-type Ca2+ (Cav3) have been linked to spinal hyperexcitability in both injury and pain (Hains et al. 2003a; Heinke et al. 2004a). Most of these same channels have also been implicated in persistent inward currents (PICs) and plateau potentials in both motoneurons and dorsal horn neurons (Hains et al. 2003a; Heckman et al. 2003; Hounsgaard and Kiehn 1989; Morisset and Nagy 1999). Where most studies have been carried out in motoneurons, plateau potentials have been reported in small subpopulations of neurons in the substantia gelatinosa (Yoshimura and Jessell 1989) and deep dorsal horn (Derjean et al. 2003; Monteiro et al. 2006; Morisset and Nagy 1999; Russo and Hounsgaard 1996).
PICs with Na+ and/or Ca2+ components have been reported in a larger fraction of lamina I (Prescott and De Koninck 2005) and laminae II–IV neurons (Murase et al. 1986). PICs, particularly in the dorsal horn, are modulated by a number of intrinsic spinal and descending supraspinal transmitters including substance P, GABA, glutamate, 5-HT, and acetylcholine (Derjean et al. 2003; Herrero et al. 2000; Murase et al. 1986; Russo et al. 1997, 1998). PICs generating plateaus become more readily activated and more sensitive to modulation in motoneurons in the chronically transected cord (Harvey et al. 2006; Li and Bennett 2003), but the actions of PICs in dorsal horn neurons after chronic cord transection remain unstudied.
Recently, the GAD67-GFP transgenic mouse has proven useful in examinations of spinal GABAergic neurons (Dougherty et al. 2005; Heinke et al. 2004b). We have shown that GAD67-GFP identified lamina I GABAergic neurons divide into at least two different populations of neurons based on their firing properties (Dougherty et al. 2005). Given the importance of GABAergic neurons to the inhibitory control of excess spinal cord excitability after SCI, the main objective of the present study was to determine the effects of complete spinal cord transection on lamina I GABAergic membrane properties, focusing on changes in active cellular properties.
Some results have been presented in abstract form (Dougherty and Hochman 2005, 2006).
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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). Some lamina I neurons may have been excluded from consideration because lamina I is thicker in the central part of the cord. Spinal cord transection
Mice, postnatal day (P) 6–8, were anesthetized with 10% urethan (1.5 mg/kg). Following dorsal laminectomy to expose a segment of the thoracic spinal cord, one segment between T8 and T11 was removed. Gel foam was used to maintain the separation between rostral and caudal parts of the cord. Mice recovered for 6–9 days (mean = 8) before cell counting or electrophysiology experiments at P13–17 (mean = 15).
Surface area and cell counts
Mice at postnatal day (P) 14 were anesthetized with urethane (2 mg/kg ip) and perfused with 1:3 volume/body weight ice-cold 0.9% NaCl-0.1% NaNO2, 1 unit/ml heparin, followed by equal volume/body weight of 4% paraformaldehyde or modified Lana's fixative (4% paraformaldehyde, 0.2% picric acid, 0.16 M PO3); pH 6.9. All spinal cords were isolated and postfixed 1 h, cryoprotected in 10% sucrose, 0.1 M PO3, pH 7.4 until sectioned in 10-µm-thick slices on a cryostat (Leitz 1720).
Ten nonconsecutive sections (100 µm apart) from lumbar segments 1–3 of three control mice and three chronic SCI mice were used for comparison of surface area and lamina I cell numbers using the Neurolucida image analysis system (MicroBrightField, Williston, VT). All cell counts can only be regarded as estimates since stereological techniques were not used. This should not affect comparisons between treatment groups.
Electrophysiology
Control and chronic SCI mice (P13-17) were anesthetized with urethan (2 mg/kg ip) and decapitated, and the spinal cord was carefully dissected out of the body cavity and placed in a cooled (<4°C) artificial cerebrospinal fluid (ACSF) containing (in mM) 250 sucrose, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 glucose, 1.25 NaH2PO4, and 26 NaHCO3 at a pH of 7.4. The ACSF was continuously oxygenated with 95% O2-5% CO2. Transverse (150–250 µm) spinal slices were cut from lumbar cord using a vibrating blade microtome (Leica VT1000 S). Slices were left to recover at room temperature for 
1 h prior to the onset of experimentation. The slicing procedure was common to both groups and represents an acute spinal injury. Because an acute injury is implicit to these procedures, for simplicity we refer to the acutely injured group as the controls and to the mice with a chronic spinal transection followed by the acute injury as the SCI or spinal cord transected mice.
Patch electrodes were prepared from 1.5 mm OD capillary tubes (World Precision Instruments, Sarasota, FL) using a two-stage puller (Narishige PP83) to produce resistance values ranging from 5 to 8 M
. For the majority of the experiments, the intracellular recording solution contained (in mM) 140 K-gluconate or KF, 11 EGTA, 10 HEPES, 1 CaCl2, and 35 KOH, pH 7.3. In some electrodes, a support solution of 4 mM ATP and 1 mM GTP was also included. No differences in properties were observed between electrodes containing or not containing support solution. For some voltage-clamp experiments, intracellular recording solution contained (in mM) 120 CsF, 10 EGTA, 10 HEPES, 10 CsCl2, and 35 CsOH.
The recording chamber was continuously perfused with oxygenated ACSF (in mM: 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 glucose, 1.25 NaH2PO4, and 26 NaHCO3 at a pH of 7.4) at a rate of 2 ml/min. In some voltage-clamp experiments, the K+ channel blockers 4-aminopyridine (4-AP; 2 mM) and tetraethylammonium (TEA; 10 mM) were included in the perfusion ACSF (as noted in RESULTS).
Whole cell patch-clamp recordings were undertaken at room temperature using the Multiclamp amplifier (Axon Instruments; Union City, CA) filtered at 5 kHz (4-pole low-pass Bessel). GFP+ lamina I interneurons were identified using epifluorescent illumination. Position of the cell in lamina I was verified using differential-interference contrast optics (DIC) at x40 to show that the cell was in or adjacent to the white matter in that focal plane (Chen and Gu 2005). Then the electrode was lowered into the slice and the cell targeted for whole cell patch-clamp recordings using DIC. Both voltage- and current-clamp data were acquired on computer with the pCLAMP acquisition software Clampex (v 9.2; Molecular Devices; Union City, CA).
Immediately following rupture of the cell membrane (in voltage clamp at –80 mV), the current-clamp recording configuration was used to determine resting membrane potential (except when CsF electrodes used). Series resistance was subtracted. Experiments were conducted in both current- and voltage-clamp modes. In current clamp, cells were brought to –80 mV by injecting bias current through the headstage. Then a series of 1-s hyperpolarizing and depolarizing current steps were undertaken. Liquid junction potentials were not corrected for. Firing type was determined by the response to current steps at and above threshold (Prescott and De Koninck 2002; Ruscheweyh and Sandkuhler 2002). Membrane properties were measured and calculated as previously described (Dougherty et al. 2005). Peak inward currents were the maximum negative values measured during the first 20 ms of 100-ms voltage steps (–130 to +70 mV) from a holding potential of –80 mV. Outward currents were measured as the average current during the last 20 ms of the highest voltage step in the same protocol. Voltage ramps (100 mV, 10 or 20 mV/s) starting at –110 mV were used to examine PICs.
In some experiments, channel blockers were applied using a local perfusion system (SF-77B Perfusion Fast-Step; Warner Instrument, Hamden, CT). The following channel blockers were used (all from Sigma unless stated otherwise): tetrodotoxin (TTX; voltage-gated Na+; 1 µM), riluzole (persistent Na+; 10 µM; Tocris), CdCl2 (nonspecific Ca2+; 400 µM), NiCl2 (T-type Ca2+; 100 µM), verapamil (L-type Ca2+; 50 µM), nifedipine (L-type Ca2+; 50 µM). Leak subtraction was performed on all current responses to voltage ramps using Clampfit (Kuo et al. 2005). Resistance was measured from the initial portion of the response where the slope was constant (1st 1.0–1.3 s).
A total of 162 lamina I GFP+ cells from 57 control mice and 90 cells from 23 SCI mice with resting potentials more negative than –40 mV were included in the analysis. However, for comparison of cell membrane properties other than resting potentials, only cells with a membrane potential more negative than –50 mV were considered. Statistical comparisons between firing properties were made using a Student's t-test and reported as means ± SE. When distributions were not normal, a Mann-Whitney test was used. When distributions had unequal SDs, a Welch correction was used (InStat, GraphPad Software, San Diego, CA).
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RESULTS |
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Comparisons of thoracic spinal cord transections performed in neonate, weanling, and adult rodents have been previously described in great detail (Stelzner et al. 1975, 1979). Consistent with these reports, we observed hyperreflexive responses to develop in all mice within the first 5 postoperative days and weight support in some mice 7–8 days after spinal cord transection. Some mice did show stepping motions with their hindlimbs, particularly when moving over cage bedding (Guertin 2005; Stelzner et al. 1979). There were no obvious correlations between behavior at the time of the experiment and results from cell recordings.
By 1 wk following spinal cord transection, white matter tracts were noticeably reduced (Fig. 1, A and B). Overall there was not a significant change in the surface area of the gray matter following spinal transection. However, measured ratios of white matter area to whole cord area demonstrated that white matter tracts had significantly degenerated in the SCI mice and constituted a smaller proportion of the cord area (32 ± 2%) after the week post injury as compared with the controls (42 ± 2%; P < 0.05) (cf. Anelli et al. 2007).
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Incidence of firing properties
In cells from both control and SCI mice, three firing properties were seen in GFP+ lamina I neurons –tonic, initial burst, and single spike (Dougherty et al. 2005). The incidence of firing properties encountered were not statistically different between cells from control and spinal transected mice (P = 0.45 Fisher's exact test). Tonically firing cells made up 34% of the control population and 42% of the population from SCI mice. Initial burst cells were encountered in 28 and 27% of cells from control and SCI mice, respectively. Thirty-four percent of control cells and 26% of cells from SCI mice were single spike. The remaining cells were unclassified (4% control, 6% SCI).
Cellular properties
Resting membrane potentials were compared between the control and SCI population (Fig. 2). When binned in 5-mV intervals and plotted as percent incidence, cells from both control and spinal transected mice show apparent bimodal distributions. The more depolarized peak is at –40 mV in both. However, the more hyperpolarized peak is 5 mV more hyperpolarized in cells from controls than in cells from SCI mice, suggesting that a subpopulation of neurons have undergone a depolarizing shift in their resting membrane potential. Shifting the control peak by 5 mV toward 0 aligns the distributions (Fig. 2, dashed line).
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). For any two measured membrane properties, there were no overall differences in regression coefficients between cells from control and SCI mice or any evidence of changed distributions in neuron subpopulations based on firing type (data not shown). For example, in cells from control and SCI mice there was the same positive correlation between input resistance and membrane time constant (r = 0.56 in control and r = 0.59 in SCI mice), negative correlation between rheobase and membrane time constant (r = –0.72 and r = –0.70, in control and SCI mice, respectively), and lack of correlation between resistance and membrane potential (r = 0.15 in control, r = 0.11 in SCI mice). Spinal transection-induced changes based on firing type
Single spike cells were unchanged in all properties measured in the cells from SCI mice except action potential decay slope (Fig. 3). We previously reported that the measured membrane properties of tonic and initial burst cells were indistinguishable in controls (Dougherty et al. 2005). When grouping these neuron populations, membrane potential, steady-state outward current, spike height, and action potential decay slope were all altered in cells from SCI mice. Unlike controls, the membrane properties of tonic and initial burst firing types differed within the cell population from SCI mice. Rheobase was lower (49 vs. 74 pA; P < 0.01) and maximum firing frequency was higher (54 vs. 32 Hz; P < 0.0001) in cells firing tonically. Moreover, the tonically firing cells from SCI mice also had a higher maximum firing frequency than the control tonic cells (43 Hz; P < 0.05).
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A subset of cells from spinal transected mice had noticeably higher firing rates than control cells (Fig. 4, A and B). Additionally, in two cells from SCI mice, plateau potentials were evident around rheobase (Fig. 4C). These plateaus were never seen during current step protocols in controls.
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30 and 115 pA (Fig. 5C). Interestingly, separation of neurons according to tonic and initial burst firing shows that it is the tonic cells that fired with higher frequencies following spinal transection (Fig. 5D; black and white lines, respectively, in Fig. 5, A and B).
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Evidence of spontaneous plateau potentials was seen in few cells from control mice (7%). However, 27% of cells from SCI mice displayed at least one spontaneous plateau potential during continuous recordings. Plateaus were most commonly seen in tonically firing cells. Their duration ranged from short (50 ms) to very long duration (50 s) although most (75%) were 
1 s in duration. Plateaus spontaneously occurred within a membrane potential range of –120 to –40 mV with values ranging from –80 to –15 mV. In a recording from a particularly active cell from a spinal transected mouse (Fig. 6), the heterogeneity of plateau duration and magnitude is obvious. The increased incidence of spontaneous plateaus suggests there may be an increase in the expression of PICs, which would also contribute to the higher firing frequencies seen with f-I curves (Kuo et al. 2006).
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PICs were examined using triangular voltage ramps (–110 to –10 mV) over 5 or 10 s. PICs were not evident in most neurons in control mice (–11 ± 4 pA; Fig. 7A). However, following blockade of voltage-gated K+ channels with intracellular Cs and extracellular 4-AP and TEA, PICs were seen in all cells tested (n = 27, Fig. 7B). For the rising phase, PIC amplitudes were –38 ± 5 pA in 5-s ramps and approximately double in 10-s ramps (–75 ± 20 pA), and the corresponding falling phase values were –48 ± 6 and –104 ± 22 pA in 5- and 10-s ramps, respectively. The membrane potentials at the peak amplitudes during rise (–40 ± 2 mV) and falling phases (–46 ± 3 mV) were comparable.
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The Ca2+ channel blocker Cd2+ blocked most of the rising phase of the ramp current (96%) and partially blocked the falling phase (left 39% of the control remaining; n = 2). Ni2+, a T-type Ca2+ channel blocker (n = 2), had no effect on either rising or falling phase PIC amplitude. The L-type Ca2+ channel blockers, nifedipine and verapamil, reduced both rising and falling phases of the voltage ramps. Nifedipine (n = 7) reduced the PIC to 49% of control values on rising and 68% on the falling phase (Fig. 7). Verapamil had weaker actions. In two of three cells tested, verapamil reduced PIC amplitude to 87 and 77% of control rising and falling phase values, respectively. In summary, both voltage-gated Na+ and Ca2+ currents contribute to PICs.
While pharmacological characterization of the conductances underlying PICs in neurons in SCI mice was not performed, under normal recording conditions, the responses to voltage ramps were not significantly different from controls (n = 32; Fig. 8A1). However, in 28% of these cells, a high-threshold inward current is activated and can be seen in the falling (repolarizing) phase of the voltage-clamp ramp (Fig. 8A2). In these cells, the falling phase of the ramps had larger peak amplitudes than controls (–30 vs. –11 pA: P < 0.05). In long voltage step protocols (5 s), a high-threshold current with delayed activation and no inactivation was seen (Fig. 8B). These currents activated at approximately –30 mV, and maximum current varied but was usually 100–250 pA. None of these cells showed either accelerated firing during current steps, a sustained depolarization beyond current steps (Fig. 8C), or enhanced firing on the repolarizing side of a current ramp (Fig. 8D).
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DISCUSSION |
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Effects of injury on the spinal GABAergic system
Approximately 2/3 of SCI patients suffer from chronic pain (Finnerup and Jensen 2004), and numerous studies have demonstrated a hyper-responsive sensory apparatus after SCI (Bennett et al. 1999; Drew et al. 2001; Hains et al. 2003b; Hao et al. 2004; Zhang et al. 2005). GABAergic control of spinal excitability becomes critical after the loss of descending inhibitory controls (Millan 2002), and dysfunction in spinal inhibitory systems has been implicated in altered nociceptive activity (Baba et al. 2003; Coull et al. 2003; Drew et al. 2004; Hao et al. 1992; Moore et al. 2002; Stiller et al. 1996). Caudal to chronic cord transection, GAD-67 expression increases (Tillakaratne et al. 2000) as does afferent-evoked GABAergic synaptic actions (Garraway and Mendell 2007). These processes are likely highly dynamic as increased GAD67 expression can be normalized by exercise training (Tillakaratne et al. 2002). Here, in lamina I, there was no difference in the number, apparent distribution, or incidence of functional subpopulations of EGFP+ neurons 8 days postspinalization.
Excitability increases in a subset of GABAergic neurons following spinal transection
Interestingly, changes in cellular properties following spinalization were largely restricted to a cell subpopulation of lamina I GABAergic interneurons, those that respond to depolarizing currents steps with tonic and initial burst firing. The observed changes all appear to function to increase cell excitability. In comparison, no changes were seen in membrane properties of lamina II GABAergic neurons following chronic constriction nerve injury (Schoffnegger et al. 2006). One of the more striking observations was a depolarizing shift in the membrane potentials of cells from SCI mice. It is possible that an injury-related change in the Cl– gradient (Coull et al. 2003) may contribute to this. Because voltage threshold and rheobase were unchanged, synaptic and excitatory neuromodulatory events would be much more likely to recruit these neurons. Additionally, observed increases in spike height and outward current could contribute to an increase in excitability. An increase in spike height may be due to changes in voltage-gated Na+ or K+ channels (Chen et al. 1996; Kang et al. 2000; Melnick et al. 2004). The increase in spike heights is likely due to a decrease or change in K+ channel properties. Because the rising slope of the spike and peak inward currents remained unchanged while action potential decay was faster, K+ channels responsible for fast repolarization (A-current) are implicated (Chen et al. 1996; Kang et al. 2000). Alternatively, this could be due to changes in the kinetics of Na+ channels or a shift in the voltage dependence due to changes in auxiliary subunits (McCormick et al. 1999; Meadows et al. 2002). The increase in peak steady-state outward currents following spinal transection suggests that one of the delayed rectifier K+ channels is facilitated and is consistent with faster firing frequencies (Melnick et al. 2004). Also even though the changes were not significant, voltage thresholds of cells from SCI mice tended to be more hyperpolarized.
Spinal cord transection causes firing frequencies of tonic cells to increase
This is the first study to examine the effects of cord transection on lamina I GABAergic neurons, and we show evidence of an increased firing frequency in a subset of tonic cells. Increased spinal excitability and increases in cell firing rates have been reported following cord injury (Hains et al. 2003a; Millan 2002) and in pain models (Finnerup and Jensen 2004; Pitcher and Henry 2000), and a loss of inhibitory function has been implicated in pain following injury (Burchiel and Hsu 2001; Wiesenfeld-Hallin et al. 1997) and pain in general (Malan et al. 2002; Millan 1999; Zeilhofer 2005). While there have been no studies directly examining changes in the excitability of inhibitory neurons, based on the aforementioned studies, one would predict reduced excitability. On the other hand, following the loss of descending inhibition, compensatory homeostatic mechanisms intrinsic to the spinal cord may warrant the increases as seen here, in GAD-67 expression (Tillakaratne et al. 2000) and in afferent-evoked GABAergic synaptic actions (Garraway and Mendell 2007).
Increases in firing frequency were specific to tonic firing cells, presumably to result in stronger inhibitory actions on spinal circuits. Because only 11% of lamina I cells are local interneurons (Bice and Beal 1997), and GABAergic neurons comprise 1/4 of the lamina I population (Polgar et al. 2003; Todd and Spike 1993), some GABAergic cells must be propriospinal neurons. Most tonic and initial burst cells are the larger fusiform cells rather than small multipolar, single spike cells (Dougherty et al. 2005). Therefore tonic cells may be propriospinal neurons with more widespread projections. Hence their lesion-induced excitability increase may exert more widespread compensatory inhibitory actions. In contrast, single spike cells may be local circuit interneurons and less relevant to the control of and compensatory response to overall spinal hyperexcitability.
Spontaneous plateaus are more frequent and PICs are evident in cells from SCI mice
As seen in unidentified neurons in the substantia gelatinosa (Yoshimura and Jessell 1989) and deep dorsal horn (Derjean et al. 2003; Monteiro et al. 2006), only a small proportion of lamina I GABAergic neurons have spontaneous plateau potentials. However, a greater number of cells displayed plateaus following chronic cord injury. Plateau potentials depend on neuromodulation in motoneurons (Hounsgaard and Kiehn 1985). Interestingly, plateau potentials disappear after acute cord transection but re-emerge much more prominently in chronically spinal transected rodents (Li and Bennett 2003) and appear to be one mechanism that results in hyperreflexia and spasticity (Bennett et al. 2004).
PICs underlie the plateaus in motor (Carlin et al. 2000; Heckman et al. 2003; Hounsgaard and Kiehn 1989; Li and Bennett 2003), deep dorsal horn (Morisset and Nagy 1999), and ventral horn neurons (Theiss et al. 2007). In particular, L-type Ca2+ currents and persistent Na+ currents are responsible, both in normal (Hounsgaard and Kiehn 1989; Kuo et al. 2005) and in chronically transected (Li and Bennett 2003) animals. Both persistent Na+ and persistent Ca2+ currents have been demonstrated in lamina I tonically firing cells where they amplify and prolong depolarization (Prescott and De Koninck 2005).
PICs were not found in normal recording conditions in most cells from control or SCI mice. Following K+ current blockade with Cs+ (and in some cases 4-AP and TEA), persistent currents were seen in all cells tested. Although firing properties could not be measured in these cells, it is likely that PICs are present in all GABAergic cells in lamina I, regardless of firing type. This conflicts with reports that only tonic cells in lamina I have PICs (Prescott and De Koninck 2005). It is possible that differences in the protocols used to evoke the PICs, differences in the recording conditions, or bias toward excitatory cell types may be responsible for the conflicting results. Because the currents were evident only after K+ channel blockade, it is likely that the channels underlying these currents are on the distal dendrites (Carlin et al. 2000; Hounsgaard and Kiehn 1993; Russo and Hounsgaard 1996). Both persistent Na+ channel blockers and L-type Ca2+ channel blockers were effective at reducing the currents. A model of PICs in tonic firing lamina I cells predicts the activation of the Ca2+ current to be dependent on the persistent Na+ current and that the maintenance of the Na+ current will depend on the Ca2+ current activation (Prescott and De Koninck 2005). Here both TTX and Cd2+ individually are capable of eliminating most of the PICs, suggesting that these currents are largely interdependent to generate PICs.
After spinal transection, a high-threshold current with a delayed activation emerges in a subset of cells. It is possible that this current is required for the spontaneous plateaus seen since they are expressed in the same percentage of cells (28 and 27%, respectively). This current is similar to dendritic L-type calcium currents reported in other regions (Carlin et al. 2000; Mermelstein et al. 2000) and has threshold properties consistent with L-type Ca2+ channels (Huang 1989). Self-sustained depolarizations at the offset of current steps and increased firing frequencies during the hyperpolarizing phase of ramps are properties characteristic of cells which generate plateaus. Despite the increased incidence of plateaus and PICs in cells from spinal cord transects, none of these cells displayed self-sustained depolarizations or increased firing frequencies. It is possible that the PICs would become larger at longer durations after spinal cord transection (Li and Bennett 2003). Because the activation of these currents is slow, it is possible that their activation is too delayed to affect spiking during a current ramp. Additionally, the long somatic current steps may not have been depolarizing enough to activate these currents out in the dendrites, possibly explaining the lack of self-sustained depolarization.
Conclusion
In a subset of lamina I GABAergic neurons, spinal transection induces a more depolarized resting potential and increased firing frequencies and plateau potentials, supporting a resultant increase in neuronal excitability. The mechanisms responsible for altering excitability remain unknown. Because descending modulatory actions are predominantly inhibitory to dorsal horn (Millan 2002) and their actions are lost after chronic cord transection (see Hadjiconstantinou et al. 1984), an increased GABAergic neuronal excitability may result as part of a compensatory homeostatic response, albeit insufficient.
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ACKNOWLEDGMENTS |
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Present address for K. J. Dougherty: Dept. of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Hochman, Whitehead Biomedical Research Bldg., Rm. 644, Emory University School of Medicine, 615 Michael St., Atlanta GA 30322 (E-mail: shochm2{at}emory.edu)
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, control means; , SCI means. Data are presented as means ± SE. *, statistical significance (P < 0.05). A: following spinal transection, tonic/initial burst cells had more depolarized membrane potentials, larger steady-state outward currents, and larger spike heights. B: the other passive properties measured (resistance, 
m, and cell capacitance) remained unchanged. C: voltage threshold, rheobase, peak inward current, and overshoot were statistically unchanged. While the rise slope was unchanged, the decay slope was less steep in cells from spinal transects.
