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Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, MB, Canada R3E 3J7
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(Received 17 March 2004;
accepted after revision 26 April 2004;
first published online 30 April 2004)
Corresponding author B. Fedirchuk: Department of Physiology, University of Manitoba, 730 William Avenue, Winnipeg, MB, Canada R3E 3J7. Email: brent{at}scrc.umanitoba.ca
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Introduction |
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Since this initial description, we have conducted computer modelling studies that have examined putative mechanisms underlying the Vth lowering seen during fictive locomotion in the decerebrate cat (Dai et al. 2002). Modulation of ionic conductances within the modelled motoneurones could produce a hyperpolarization of Vth. Specifically, enhancement of the conductance or activation of the Na+ channels underlying action potentials and/or a reduction in K+ conductance could lower Vth. Since it is well established that monoamines are major neuromodulatory systems influencing the mammalian motor system (for review see Schmidt & Jordan, 2001), we hypothesized that monoamines may exert a modulatory effect on Vth. We have recently shown that bath application of serotonin (5-HT) or noradrenaline (NA) induces a rapid and reversible hyperpolarization of Vth in lumbar ventral horn neurones in the spinal cord isolated from a neonatal rat (Fedirchuk & Dai, 2004). This prior study showed that (1) Vth hyperpolarization can be also demonstrated in rat, (2) Vth hyperpolarization can be seen in neonatal animals (postnatal day 1–5), and (3) that bath applied 5-HT and NA can induce a temporary hyperpolarization of Vth in spinal neurones that is analogous to that seen during locomotion in the cat.
Despite our demonstration of a monoamine-induced Vth hyperpolarization in spinal neurones of the neonatal rat, it remains undetermined whether endogenous mechanisms of inducing hyperpolarization of Vth are functional at this developmental age, and also whether there is a hyperpolarization of Vth associated with fictive locomotion in the rat. However, the most common method used to evoke locomotor activity in neonatal rat preparations is to apply transmitters, including monoamines, to the spinal cord (Smith et al. 1988; Cazalets et al. 1992; Kiehn et al. 1992; Cowley & Schmidt, 1994a; Kiehn & Kjaerulff, 1996; Kiehn et al. 1999). Application of 5-HT or NA to evoke locomotor activity would confound experiments designed to examine a locomotor-associated hyperpolarization of Vth, because the agents applied to produce locomotion might themselves affect Vth. Therefore, we used an isolated brainstem–spinal cord neonatal rat preparation (as in Smith et al. 1988) and sought to evoke locomotor activity by electrical stimulation of the brainstem in a manner analogous to fictive locomotion in the cat. Atsuta et al. (1988) have previously reported that electrical stimulation of the medioventral medulla induced motor output that was locomotor-like (Atsuta et al. 1990) in this preparation. More recent studies have demonstrated the ability of focal electrical brainstem stimulation (Jordan & Schmidt, 2002) and gross electrical brainstem stimulation (Zaporozhets et al. 2004) to initiate locomotor-like ventral root activity in the brainstem–spinal cord isolated from a neonatal rat.
Therefore, the goals of the present study were to use the neonatal brainstem–spinal cord preparation to determine (1) whether endogenous modulatory systems are able to induce Vth hyperpolarization of motoneurones in the neonatal rat, (2), whether hyperpolarization of Vth is dependent on locomotor activity, and (3) whether hyperpolarization of Vth by endogenous pathways is mediated by monoamines. Portions of these data have been presented in preliminary form (Gilmore & Fedirchuk, 2002).
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Methods |
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All surgical procedures were developed in accordance with Canadian Council on Animal Care guidelines and were approved by the University of Manitoba animal protocol committee. Neonatal Sprague–Dawley rats (postnatal day 1–5; n= 62) were anaesthetized with halothane in a closed chamber. The animal was then moved to a dissecting dish, and a precollicular/postmammillary mechanical decerebration was rapidly performed. The thorax and abdomen were eviscerated, and the preparation was transferred to a clean Slygard-bottomed chamber and superfused with 4°C artificial cerebrospinal fluid (aCSF). The aCSF contained (mM) 125 NaCl, 2.5 KCl, 1 MgCl2, 1.25 Na2HPO4, 26 NaHCO3, 2.0 CaCl2 and 25 D-glucose, and was aerated with 95% O2–5% CO2 under pressure. Dorsal and ventral laminectomy exposed the spinal cord and brainstem, and the dura mater covering the medulla and spinal cord was removed. All spinal roots were cut near the dorsal root ganglia and the brainstem–spinal cord was transferred to a Sylgard-bottomed recording chamber and pinned ventral side up with 0.1 mm insect pins, The preparation was slowly warmed to 25°C, and the temperature monitored, and maintained with a heating lamp except where noted.
Bath partitions
In some experiments, 35 mm photographic film and Vaseline were used to divide the bath into brainstem, cervical, and thoracolumbar partitions. This allowed (1) selective cooling of the cervical spinal cord with 4°C aCSF, or (2) administration of pharmacological agents to the spinal cord without influencing the brainstem. During cooling of the cervical partition, the temperatures of the thoracolumbar and brainstem partitions were monitored and maintained by washing with 25°C aCSF.
Electrical brainstem stimulation and ventral root recording
Electrical stimulation of the brainstem was done with a monopolar tungsten electrode (10–100 k
; Microprobe, Inc. Carlsbad, CA, USA) placed on the surface of the ventromedial medulla, 1.5–2.0 mm lateral to midline and 1.0–2.0 mm caudal to the level of decerebration. The optimal site for evoking rhythmic ventral root (VR) activity was determined by repositioning the brainstem stimulating electrode in 0.25 mm increments until stable rhythmic activity was obtained. Electrical stimulation was done using a custom-built constant current stimulator delivering 0.5–5.0 ms square pulses, 200 µA to 10 mA in amplitude, delivered at 1–3 Hz.
Ventral root (VR) recordings from the second and fifth lumbar ventral roots on both the left and right sides (lL2, lL5, rL2, rL5) were obtained using custom-made plastic suction electrodes. VR records were continuously monitored for motor output during brainstem stimulation, and were digitized at 5 kHz using a PC-based data acquisition and analysis platform. Previous work has shown that L2 and L5 VR activity largely corresponds to activity in flexor and extensor motoneurone pools, respectively (Cowley & Schmidt, 1994b; Kiehn & Kjaerulff, 1996). However, we acknowledge that caution must be used when ascribing patterns of VR activity to motor behaviours like locomotion (see Cowley & Schmidt, 1994b). In the present study, we categorized the VR activity produced by electrical stimulation of the brainstem as follows: (1) ‘locomotor-like’ activity was defined as rhythmic alternating activity of left and right L2 VRs with appropriate rhythmic alternating activity of at least one L5 VR and was considered to be consistent with the VR pattern expected during fictive locomotion; (2) ‘ipsilateral/contralateral’ (ipsi/contra) alternation was rhythmic activity alternating between left and right L2 VRs, without rhythmic alternating activity in either L5VR; (3) ‘ipsilateral/ipsilateral’ (ipsi/ipsi) alternation was rhythmic activity with alternation between an L2 VR and the ipsilateral L5 VR, without alternating activity in the contralateral VRs; (4) ‘rhythmic activity’ was bursting activity not alternating with activity in other roots (it could include a single root rhythmically discharging); (5) ‘tonic activity’ was increased tonic activity in one or more roots; and (6) ‘no ventral root output’ was the failure of brainstem stimulation to elicit measurable activity in the VRs.
Single cell recordings
Ventral horn neurones of the lower lumbar segments were targeted for single-cell recording using glass microelectrodes pulled with a Narishige PP-83 two-stage puller and filled a solution containing 140 mM potassium gluconate, 0.2 mM EGTA, 10 mM Hepes, and KOH to bring the pH to 7.3. The filled electrodes had resistances ranging from 4.1 to 4.5 M. Usually, the microelectrode was introduced into the ventral horn from the ventral surface of the spinal cord; however, in a few experiments, a short midline hemisection was done so that the electrode could be introduced from the medial surface of the ventral horn (as done in Fedirchuk & Dai, 2004). A whole-cell single cell recording arrangement was obtained using the ‘blind patch’ technique (Blanton et al. 1989). An Axopatch 1D microelectrode amplifier controlled with pCLAMP 7 software (Axon Instruments) was used for recording. Series resistance was monitored, was usually < 45 M, and was uncompensated. Tip potentials were not compensated.
Antidromic stimulation
In 29 experiments electrical stimulation of the VR (300–600 µA) corresponding to the spinal segment containing the neurone being recorded was done in the attempt to antidromically activate the neurone. Antidromically activated ventral horn neurones are considered motoneurones. Neurones which either were not activated by VR stimulation, or were recorded in preparations in which VR stimulation was not attempted are considered unidentified ventral horn neurones.
Measurement of Vth
Vth was determined using a voltage clamp protocol as has been done previously (Fedirchuk & Dai, 2004). An initial holding potential of –60 mV was used to approximate the resting membrane potential, and 100 ms depolarizing steps that increased in 2 mV increments were applied. Steps were delivered at a repetition rate of 2 Hz. Large amplitude fast inward currents which would have mediated action potentials were evident on the recorded current trace, and the potential of the smallest depolarizing step capable of inducing a fast inward current was considered to be Vth. Vth values determined in this way were stable for prolonged periods in the absence of brainstem stimulation. A hyperpolarization of Vth is manifest as a smaller depolarizing voltage step (i.e. more negative membrane potential) being able to induce a fast inward current.
Antagonists
In some experiments, the 5-HT2 antagonist ketanserin (Sigma–Aldrich, Oakville, ON, Canada) (2–20 µM) was added to the thoracolumbar bath partition. The effects of electrical stimulation of brainstem on the Vth of a neurone could then be compared prior to (control), and in the presence of, bath-applied ketanserin. Identical parameters of brainstem stimulation were used in both cases.
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Results |
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Electrical stimulation of the surface of the ventromedial medulla can induce an alternating pattern of VR that is consistent with locomotor-like activity (e.g. Fig. 1A). The locomotor-like VR activity typically had a cycle frequency of 0.3–0.5 Hz, which is consistent with frequencies reported during chemically evoked locomotor-like activity (Cowley & Schmidt, 1994a, Cazalets et al. 1992), and the frequency was somewhat dependant on the frequency of stimulation (1–3 Hz, not illustrated). To ensure that the locomotor-like VR activity was produced by activation of the brainstem rather than current spread that might have directly activated spinal locomotor circuitry, in six experiments (not illustrated) the cervical bath partition was cooled to 4°C to block action potential propagation (see Castro-Moure & Goshgarian, 1996). In all cases cooling the cervical cord blocked the brainstem-evoked locomotor-like VR activity and re-warming the cervical cord restored the locomotor-like activity. Therefore, locomotor-like VR activity was produced by neuronal fibres descending from the brainstem rather than from spread of the stimulating current to the thoracolumbar spinal cord.
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Figure 1 shows a representative example of an antidromically identified lumbar motoneurone recorded prior to and during brainstem-evoked locomotor-like VR activity. Figure 1A shows the VR root records and the rapid onset of locomotor-like activity with the onset of electrical stimulation of the brainstem (10 mA, 5 ms pulses, 2 Hz). Figure 1B illustrates the membrane current and voltage command traces for a motoneurone simultaneously recorded during the time period shown in Fig. 1A. Prior to brainstem stimulation (control, left traces), a step to –34 mV from the initial –60 mV holding potential was required to elicit a fast inward current (therefore, Vth=–34 mV). During the brainstem-evoked locomotor-like VR activity (Fig. 1B, right traces) a depolarizing step to –38 mV was sufficient to evoke a fast inward current. This represents a –4 mV change (a 4 mV hyperpolarization) of the threshold potential required to elicit the fast inward current that would underlie action potential production. Figure 1C summarizes the results from all 11 neurones recorded during brainstem-evoked locomotor-like activity. The absolute values of Vth are shown prior to and during electrical stimulation of the brainstem in the upper plot of Fig. 1C, and the values for an individual neurone are connected by a line. The relative change in Vth for each of these neurones is re-plotted (Fig. 1C, lower plot). These results show that 10/11 neurones exhibited a hyperpolarization of Vth during brainstem-evoked locomotor-like VR activity, which ranged from –2 to –18 mV (mean –6 mV), that was significant (paired t test, P= 0.004). Recovery following brainstem stimulation to the control Vth value was confirmed in 9 of these 10 neurones and occurred within 1 min (n= 8) or within 1–2 min (n= 1). These results are analogous to the previous description of a hyperpolarization of Vth in spinal motoneurones during brainstem-evoked fictive locomotion in the decerebrate cat (Krawitz et al. 2001).
Five of the 11 neurones recorded during brainstem-evoked locomotor-like activity (dashed lines in Fig. 1C) were antidromically activated by VR stimulation, and therefore identified as lumbar motoneurones. Four out of five of these motoneurones showed a hyperpolarization of Vth during the locomotor-like VR activity, and the remaining motoneurone is the single cell in which there was no change in Vth (cell 1 in Fig. 1C lower plot). Since both motoneurones and unidentified ventral horn neurones exhibited similar responses, this data is pooled in the summaries that follow; however, comparison of the overall sample of motoneurones versus unidentified cells is discussed below.
Hyperpolarization of Vth is not dependent on locomotor-like activity
Despite the ability of electrical stimulation of the brainstem to evoke a pattern of VR activity consistent with locomotor-like activity (Fig. 1A), it more often evoked other patterns of VR activity. Vth values obtained from 36 neurones were grouped according to the pattern of brainstem-evoked VR discharge, and are summarized in Fig. 2. Figure 2A shows the absolute Vth values prior to and during electrical stimulation of the brainstem. Values from individual neurones are connected by a line. Figure 2B shows the data from the corresponding plot in Fig. 2A, and the data are re-plotted as the change in Vth seen during electrical brainstem stimulation. Note that 32/36 neurones showed a hyperpolarization of Vth (mean –6 mV; range –2 to –20 mV), and hyperpolarization of Vth was evident during all patterns of rhythmic VR activity, as well as during tonic activity evoked by the brainstem stimulation. The Vth change seen during brainstem stimulation was statistically significant (each group considered individually, paired t test P < 0.05).
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In 45 neurones the recovery of Vth to the control value was confirmed following cessation of brainstem stimulation. Recovery of Vth was seen in < 1 min (n= 26), in 1–2 min (n= 9), 2–3 min (n= 3), in 3–5 min (n= 5), or in 5–10 min (n= 3). Therefore the brainstem-evoked hyperpolarization of Vth was temporary and had a time course of recovery that was slower than its onset.
Comparison of motoneurones versus unidentified ventral horn neurones
Thirty-six of 71 neurones recorded in this study were antidromically identified lumbar motoneurones. Of these one showed a depolarization of Vth during brainstem stimulation (2 mV), two showed no change of Vth during brainstem stimulation and in 33 Vth hyperpolarized during brainstem stimulation (mean –4 mV, range –2 to –14 mV). The remaining 35/71 neurones were unidentified ventral horn neurones. Two showed a depolarization of Vth (2 mV), two had no change in Vth, and 31 showed a hyperpolarization of Vth during brainstem stimulation (mean –6 mV, range –2 to –20 mV). There was no difference in the incidence, or amplitude (Mann-Whitney rank sum test, P= 0.053) of Vth change produced by brainstem stimulation when comparing motoneurones to unidentified lumbar ventral horn neurones.
Hyperpolarization of Vth can be induced by brainstem stimulation that is subthreshold for rhythmic motor-output
In order to maintain a consistent brainstem stimulation between trials, in most experiments the brainstem stimulation parameters which at the start of the experiment were able to evoke locomotor-like or alternating VR activity were used throughout the experiment. However, Fig. 4 shows VR records during a period when bouts of brainstem stimulation of increasing intensity were successively applied (denoted by bars below rL5). Simultaneously, a motoneurone was recorded, and its Vth assessed. At 100 µA, only shock artifacts were evident on the VR records, and there was no change in Vth during the stimulation (it remained at the control value of –50 mV). Increasing the stimulus intensity 200 µA induced a –2 mV hyperpolarization of Vth, but no VR activity. Tonic ventral root activity started to be evoked at 300 µA (see lL5), but no further change in Vth was seen. At 400 µA rhythmic VR activity was evident, with an ipsi/contra alternating pattern of VR activity emerging at 500 µA. Stimulation at 600 µA (not illustrated) produced the same VR activity and Vth change as 500 µA. This example shows that by grading the intensity of electrical stimulation of the brainstem, it was possible to demonstrate the differential ability of brainstem stimulation to evoke Vth hyperpolarization and VR activity. Similar effects of grading the stimulus intensity were observed in three neurones.
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Figure 5A (upper trace) shows a voltage clamp recording from a neurone during electrical stimulation of the brainstem that induced a –2 mV change in Vth (control not show). The rhythmic VR activity induced by the brainstem stimulation (600 µA, 5 ms, 1 Hz) is shown below the voltage clamp trace. Figure 5B shows that cooling of the cervical partition to 4°C blocked the hyperpolarization of Vth and the ventral root output induced by the brainstem stimulation. Figure 5C shows recovery of the brainstem-evoked VR activity and Vth hyperpolarization when the cervical partition was warmed to 25°C over 10 min. The ability of cooling of the cervical partition to effectively block the brainstem induced Vth hyperpolarization (mean –6 mV, range –2 to –12 mV) was seen for 6/6 experiments in which it was done.
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The ability of the 5-HT2A antagonist ketanserin (2–20 µM) to block hyperpolarization of Vth induced by brainstem stimulation was assessed for 16 trials in 11 neurones. The results are summarized in Fig. 6. As in previous figures, the change in Vth induced by brainstem stimulation is depicted by the bars, and each trail is represented by a pair of bars. The open bars represent the change in Vth produced by electrical stimulation of the brainstem prior to application of ketanserin to the thoracolumbar bath partition. The filled bars indicate the change in Vth induced by the electrical stimulation of the brainstem with the same parameters, but with ketanserin added to the thoracolumbar bath. As indicated above the bars, the trials are grouped according to the concentration of ketanserin that was added, and successive trials in individual neurones are indicated by matching symbols. Concentrations of ketanserin 
3 µM were ineffective in blocking hyperpolarization of Vth induced by brainstem stimulation; concentrations of 4 µM were somewhat effective in blocking or partially blocking the brainstem-evoked change in Vth; and concentrations 
5 µM completely blocked the hyperpolarization of Vth induced by electrical stimulation of the brainstem (n= 5). The brainstem-evoked hyperpolarization of Vth could be seen to recover following the washout of ketanserin (n= 1/1 trial).
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Comparison to Vth hyperpolarization observed in the decerebrate cat
The Vth hyperpolarization produced by electrical stimulation of the brainstem in the neonatal rat is in many ways analogous to the previous description of hyperpolarization of Vth in spinal motoneurones during brainstem-evoked fictive locomotion that has been described in the decerebrate cat (Krawitz et al. 2001). Both the brainstem-evoked Vth hyperpolarization in the present study and the hyperpolarization of Vth in spinal motoneurones during fictive locomotion in the decerebrate cat had a rapid onset with the initiation of brainstem stimulation. The Vth for the first action potential or fast inward current evoked during brainstem stimulation was hyperpolarized. Also, both in the present study and in Krawitz et al. (2001), the Vth recovered within minutes following cessation of brainstem stimulation. The incidence of Vth hyperpolarization was high and comparable in both preparations. Every motoneurone examined in the decerebrate cat displayed a hyperpolarization of Vth during fictive locomotion, and 64/71 lumbar neurones in the present study displayed a hyperpolarization of Vth during brainstem stimulation. Lastly, the magnitude of the Vth hyperpolarization was comparable in the present study (mean –6 mV; range –2 to –20 mV) to that reported during fictive locomotion in the decerebrate cat (mean –8.0 mV; range –1.8 to –26.6 mV).
The primary difference between the hyperpolarization of Vth produced by brainstem stimulation in the present study and that previously described in the decerebrate cat is that the Vth hyperpolarization in the cat was associated with fictive locomotion while in the present study Vth hyperpolarization was observed during electrical stimulation of the brainstem even in the absence of locomotor-like VR activity. This difference is probably produced by differences in the brainstem stimulation used in these two studies. In the decerebrate cat, fictive locomotion was evoked by electrical stimulation of the mesencephalic locomotor region (MLR) of the midbrain. Although the MLR is functionally defined by its ability to induce locomotor activity, it is thought that the cuneiform nucleus is the anatomical correlate. This midbrain site probably represents a relay site with connections projecting to other brainstem nuclei which give rise to a locomotor command pathway projecting to the spinal locomotor pattern generating circuitry (for review see Jordan, 1986). Therefore, the MLR in the cat provides the opportunity to selectively activate the brainstem circuitry able to induce locomotor activity. This is supported by the ability of relatively low intensity MLR stimulation (< 150 µA) to often induce fictive locomotion in decerebrate cat preparations.
In contrast to MLR stimulation, electrical stimulation of the medulla was used to evoke locomotor-like activity in the in vitro neonatal rat brainstem–spinal cord preparation in the present study. The most reliable and successful stimulation site was at the surface of ventromedial medulla, and often required high stimulus intensities (up to 2 mA). Stimulus intensities even higher than this would have probably increased our success at evoking locomotor-like activity (Zaporozhets et al. 2004). However, brainstem stimulation > 2 mA caused drift in the baseline of the single-cell recordings in the present study, and was therefore not used. The ventromedial medullary site stimulated is probably not as specific for activating brainstem locomotor circuitry as the MLR in the cat, and therefore the variable success of evoking locomotion in these preparations may not be surprising. It is notable that grading the intensity of electrical brainstem stimulation could demonstrate the ability of the stimulation to evoke Vth hyperpolarization at stimulus intensities that were subthreshold for evoking rhythmic VR output. Since we have implicated a descending serotonergic projection in the production of the Vth hyperpolarization, it appears that the ventromedial medullary stimulation site used in this study may be relatively more efficient at inducing Vth hyperpolarization than inducing locomotor output. It is also possible that a descending serotonergic projection mediates both the Vth hyperpolarization and the induction of locomotor activity, but that Vth hyperpolarization in spinal neurones is manifest prior to the induction of rhythmic activity in spinal neuronal circuits.
Potential mechanisms underlying hyperpolarization of Vth
In addition to its ability to induce locomotor activity (see Introduction), 5-HT is known to have various effects on neurones. 5-HT can produce depolarization of motoneurones (Neuman, 1985; Connell & Wallis, 1988; Elliot & Wallis, 1990; Takahashi & Berger, 1990; see also Binder et al. 1993) that is mediated by (1) the enhancement of an slow inward rectifier current carried by K+ and Na+ ions (i.e. Ih,Wang & Dun, 1990; Takahashi & Berger, 1990; Kjaerulff & Kiehn, 2001), (2) the facilitation of a low voltage-activated Ca2+ current (Berger & Takahashi, 1990), and (3) the inhibition of a fast inward rectifier current carried by K+ (IKir, Kjaerulff & Kiehn, 2001). These effects serve to reduce leak of current out of the cell, thereby enhancing neuronal excitability. 5-HT can induce a reduction of the afterhyperpolarization (AHP) following an action potential (Van Dongen et al. 1986), and can also facilitate non-linear integrative properties in motoneurones. As examples, 5-HT promotes bistable firing in spinal animals (Hounsgaard et al. 1988; Hounsgaard & Kiehn, 1989) and persistent inward currents can be enhanced by monoaminergic agents (Lee & Heckman, 1999). Serotonin also facilitates conductances mediated by NMDA receptors (MacLean et al. 1998; MacLean & Schmidt, 2001).
Despite the numerous known effects of 5-HT on neurones, the ability of 5-HT to hyperpolarize the threshold for neuronal activation has not been previously reported. Perhaps this might be due to the common use of TTX to synaptically isolate the recorded neurone, but which would also preclude examination of neuronal threshold properties. In previous computer modelling work, the putative modulatory process that was most effective in inducing a hyperpolarization of Vth without concomitant changes in action potential shape was the enhancement of the activation of, or conductance through, the fast sodium current underlying action potentials (Dai et al. 2002). The modulation of the amplitude and inactivation profile of sodium channels via phosphorylation has been documented (West et al. 1991; Cantrell et al. 1997, 1999) and a role for these modulatory processes in mediating neuronal plasticity has been suggested (see Cantrell & Catterall, 2001). It is therefore possible that similar modulatory mechanisms underlie the monoaminergic hyperpolarization of neuronal Vth observed in the present study.
As discussed in Fedirchuk & Dai (2004), it is possible that modulation of channel types other than the fast sodium channels underlying spiking might be involved in the monoamine-induced Vth hyperpolarization. Reducing a potassium conductance could also hyperpolarize Vth, although to a lesser degree than direct manipulation of sodium channels (Dai et al. 2002). In addition, persistent inward currents mediated by calcium and sodium channels are activated at membrane potentials near or even below spike threshold (Lee & Heckman, 2001; Li et al. 2004). Therefore monoaminergic facilitation of persistent inward currents (Lee & Heckman, 1999), or the NMDA current (MacLean & Schmidt, 2001), might cause a contribution of these currents to spike initiation and also contribute to Vth hyperpolarization.
The present study demonstrates a hitherto unrecognized role for descending serotonergic projections. Descending monoaminergic projections facilitate the recruitment of neurones in the spinal motor system by hyperpolarizing the membrane potential at which they are activated. The neonatal rat brainstem–spinal cord readily demonstrates this modulatory process and will be a useful preparation for the further exploration of the pharmacological and cellular mechanisms underlying this new aspect of neuromodulation.
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