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1 Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK2 Neurosciences Program, Ottawa Health Research Institute and University of Ottawa, Ottawa, Ontario, Canada, K1Y 4E9
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Abstract |
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(Received 23 September 2003;
accepted after revision 9 December 2003;
first published online 12 December 2003)
Corresponding author D. Spanswick: Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. Email: d.c.spanswick{at}warwick.ac.uk
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Introduction |
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Considerable evidence exists supporting a role for GABA as a neurotransmitter to SPNs including electrophysiological data (Backman & Henry, 1983; Inokuchi et al. 1992; Dun et al. 1993; Krupp & Feltz, 1993; Deuchars et al. 1997), electron-microscopic data (Bogan et al. 1989; Cabot et al. 1995; Llewellyn-Smith et al. 1995) and histochemical data (Bacon & Smith, 1988; Chiba & Semba, 1991; Llewellyn-Smith et al. 1995, 1997, 1998, 2002). Indeed, it has been proposed that GABAergic boutons comprise up to half of the total innervation of SPNs (Llewellyn-Smith et al. 1995, 1998). Despite the extensive literature supporting a role for GABA as a neurotransmitter to SPNs, relatively little is known of the functional roles of GABA receptors in SPNs, the receptors mediating synaptic transmission and how SPNs integrate this information to formulate output responses. Here we demonstrate the expression of functional postsynaptic GABAA and GABAB receptors and a role for GABA in the bilateral regulation of SPNs. We further demonstrate that GABA-mediated synaptic inputs can lead to activation of intrinsic conductances conducive to generation of burst firing patterns of activity. Such a mechanism could be important in entrainment of SPNs to sympathetic rhythms. Part of this work has been previously published in abstract form (Wilson & Spanswick, 1998; Whyment et al. 2000).
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Methods |
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Electrophysiological recordings were made from transverse and longitudinal thoracolumbar spinal cord slices as previously described (Logan et al. 1996). Briefly, Sprague–Dawley or Wistar Kyoto rats, aged 7–16 days (either sex), were terminally anaesthetized using 4% Enflurane in O2 (Abbott laboratories, Queensborough, Kent, UK) and decapitated, the spinal cord was removed and transverse or longitudinal sections were cut into 300–450 µm thick slices using either a Vibratome (Technical Products International Inc., St Louis, MO, USA) or Leica VT1000S (Leica Microsystems UK, Milton Keynes, UK). Slices were maintained in artificial cerebrospinal fluid (aCSF) at room temperature for 1 h after slicing before transferring to a recording chamber. For recording, individual slices were held between two grids in a custom-built chamber continuously perfused with aCSF at a rate of 4–10 ml·min-1, illuminated from below and viewed under a dissection microscope. The aCSF was of the following composition (mM): NaCl, 127; KCl, 1.9; KH2PO4, 1.2; CaCl2, 2.4; MgCl2, 1.3; NaHCO3, 26; D-glucose, 10; equilibrated with 95% O2/5% CO2.
Electrophysiological recordings
Whole cell recordings were performed at room temperature from neurones in the IML with an Axopatch 1D amplifier (Axon Instruments, Union City, CA, USA), using methods described in detail previously (Logan et al. 1996; Spanswick et al. 1998). Patch pipettes were pulled from thin-walled borosilicate glass (GC150-TF10, Clarke Electromedical, Pangbourne, Berkshire, UK) and had resistances of between 3 and 8 M
when filled with intracellular solution of the following composition (mM): potassium gluconate, 130; KCl, 10; MgCl2, 2; CaCl2, 1; EGTA-Na, 1; Hepes, 10; Na2ATP, 2; Lucifer yellow, 2 (or biocytin, 5); pH adjusted to 7.4 with KOH, osmolarity adjusted to 310 mosmol l-1 with sucrose. Series resistance compensation of approximately 70–80% was applied for whole-cell voltage clamp experiments. Correction of the liquid junctional potential was applied to whole-cell recordings and access resistance ranged between 5 and 25 M. Neuronal input resistances were measured by injecting small rectangular-wave hyperpolarizing current pulses (10–50 pA) and measuring the amplitude of resulting electrotonic potentials. Recordings were monitored on an oscilloscope (Gould 1602, Gould Instrument Systems), displayed on a chart recorder (Gould, Easygraf TA240) and stored on either VHS (Sony, SLV-E230, signals being digitized at 2 kHz (Sony, PCM-701ES)), or digital audio tapes (Biologic, DTR-1205) for later off-line analysis. In addition, data were filtered at 2–5 kHz (1 kHz for voltage clamp data), digitized at 2–10 kHz (Digidata 1200A, Axon Instruments) and stored on a PC running pCLAMP 8.2 data acquisition software. Analysis of electrophysiological data was carried out using Clampfit 8.2 software (Axon Instruments). SPNs were identified as previously described (Logan et al. 1996; Spanswick et al. 1998). Briefly, SPNs were identified according to their characteristic morphology revealed postrecording by the presence of biocytin or Lucifer yellow introduced from the recording electrode, location in the lateral horn and characteristic electrophysiological properties.
Bipolar concentric stimulating electrodes (Clark Electromedical, Pangbourne, Berkshire, UK) were used to stimulate (1–10 V, 0.2 ms, 0.03–0.1 Hz, unless otherwise stated) either the ipsi- or contra-lateral lateral funiculi (iLF and cLF, respectively). In longitudinal slices, a diagonal cut was made at the caudal end of slices to ensure correct orientation of the slice in the recording chamber and positioning of stimulating electrodes rostral to the recording electrode. The latency of synaptic responses was measured relative to the onset of the stimulus artefact.
Statistical analyses
Statistical analysis was performed using Excel 2002 (Microsoft) with all values given as means ±S.E.M. Statistical significance was determined using Student's two-tailed t tests, paired or unpaired as appropriate. P < 0.05 was taken to indicate statistical significance.
Drugs
The following drugs were used: baclofen (10–100 µM), bicuculline methiodide (1–10 µM), caesium chloride (CsCl2, 1–10 mM), 
-aminobutyric acid (GABA, 2 mM), idazoxan hydrochloride (200 nM), muscimol (10–100 µM), 6-nitro-7-sulfamoylbenzo(f)quinoxaline-2,3-dione (NBQX, 5 µM), strychnine hydrochloride (2 µM) and yohimbine hydrochloride (1 µM) (all from Sigma); cis-4-aminocrotonic acid (CACA, 200 µM), D(–)2-amino-5-phosphonopentanoic acid (D-APV, 10 µM), (RS)-3-amino-2-(4-chlorophenyl)-2-hydroxypropyl-sulphonic acid (2-hydroxysaclofen, 100–200 µM), 2-(3-carboxypropyl)-3-amino-6-(p-methoxy- phenyl)pyridazinium bromide (SR95531, 10 µM) ((2S)-3-[[(15)-1-(3,4-dichlorophenyl-0-ethyl] amino-2-hydroxy-propyl](phenylmethyl) phosphinic acid (CGP 55845, 200 nM), and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidium chloride (ZD7288, 10 µM) (all from Tocris Cookson, Bristol, UK) and tetrodotoxin (TTX, 500 nM, from Alomone Laboratories, Israel).
CGP55845 and NBQX were prepared as a stock solution using 100% DMSO (Sigma) and diluted to the required concentration in aCSF immediately prior to use, resulting in a maximal DMSO concentration of < 0.1%. All other drugs were prepared as stock solutions in distilled water and diluted to the required concentration in aCSF immediately prior to use. The drugs were administered to the slice by perfusion from 50 ml syringes arranged in line with the main aCSF reservoir by a series of three-way valves. The agonist final concentrations reported represent the concentrations within the perfusion system and take no account of dilution within the recording chamber. Antagonists were applied for at least 10 min prior to the addition of agonists to ensure complete equilibration within the recording chamber.
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Results |
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. Isolation of GABA-mediated synaptic inputs to SPNs
Electrical stimulation (0.03–0.1 Hz) of either the iLF or cLF evoked fast excitatory postsynaptic potentials (EPSPs) or excitatory postsynaptic currents (EPSCs) in all SPNs tested (n= 36). Application of the non-NMDA receptor antagonist NBQX (5 µM) and the NMDA receptor antagonist D-APV (10 µM) reversibly blocked these fast EPSPs in all neurones and unmasked fast IPSPs in 27/36 (75%) and 17/26 (65%) of SPNs following stimulation of the iLF and cLF, respectively (Fig. 1Aa). IPSPs had mean latency, 10–90% rise time and half-decay times of 2.4 ± 0.3, 8.1 ± 1.9 and 47 ± 5 ms, respectively (means ±S.E.M.), following stimulation of the iLF (data from 27 neurones, values calculated from averages of 16 consecutively evoked responses for each neurone) and 7.8 ± 1.7, 10.1 ± 1.9 and 56 ± 4 ms, respectively, following stimulation of the cLF (data from 17 neurones, values calculated from averages of 16 consecutively evoked responses for each neurone) at holding or resting potentials between -35 and -50 mV. IPSPs showed graded responses to increasing stimulus intensity (Fig. 1Ba for iLF; Fig. 1Ca for cLF.), constant rise time (Fig. 1Bb and Cb) and no failures upon repetitive stimulation (10–100 Hz, Fig. 1Bc and Cc) suggesting a monosynaptic origin.
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2 receptor antagonists idazoxan (200 nM) and/or yohimbine (1 µM) were routinely included in the applied drug solutions. Application of the GABAA receptor antagonists bicuculline (1–10 µM, n= 27; Figs 1Ab and 2) and SR95531 (10 µM, n= 4; data not shown) reversibly abolished IPSPs in a concentration-dependent manner. The threshold for bicuculline-induced complete inhibition of IPSPs or IPSCs was 5 µM (Fig. 2A). In addition, in three neurones, IPSCs evoked by stimulation of the iLF (n= 1) or cLF (n= 2) were partially suppressed upon application of the glycine receptor antagonist, strychnine (2–5 µM) and subsequently abolished by bicuculline (Fig. 2B and C). IPSPs (Fig. 3Aa) and IPSCs (Fig. 3Ba) increased in peak amplitude upon membrane depolarization and decreased and reversed polarity upon membrane hyperpolarization to potentials more negative than around -55 mV. The mean reversal potential for IPSPs and IPSCs were -57 ± 2 mV (n= 6) and -56 ± 2 mV (n= 4) for the iLF (Fig. 3Ab) and the cLF (Fig. 3Bb), respectively, close to the reversal potential for chloride ions under our recording conditions.
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The effects of GABA receptor agonists on SPN
To further clarify the properties of postsynaptic GABA receptors on SPNs, the effects of GABA receptor agonists were investigated on 49 neurones. GABA (2 mM, n= 6), the GABAA receptor agonist muscimol (10–100 µM, n= 24), the GABAB receptor agonist baclofen (10–100 µM, n= 15) or the GABAC receptor agonist CACA (200 µM, n= 4) was bath applied to the slice by superfusion for 10–120 s. Bath application of GABA induced membrane hyperpolarization in all SPNs tested. The response was characterized by hyperpolarization of the membrane from a mean resting or holding potential of -45.3 ± 1.9 mV to -52.0 ± 2.0 mV, a mean peak membrane hyperpolarization of 6.7 ± 1.6 mV (Fig. 4B). GABA-induced membrane hyperpolarization was associated with
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at rest to 250 ± 42 M in the presence of GABA, amounting to a 54.6 ± 3.9% decrease (Fig. 4B). Muscimol application produced membrane hyperpolarization in 17 cells from a mean resting or holding potential of -45.9 ± 4.9 mV to -51.9 ± 3.6 mV, a mean hyperpolarization of 6.0 ± 0.6 mV at the peak of response (Fig. 4Aa and b). A membrane depolarization was observed in seven SPNs, at resting potentials more negative than the chloride reversal potential under our recording conditions, from a mean resting potential of -60.3 ± 2.7 mV to -52.7 ± 1.9 mV, a mean depolarization of 7.6 ± 1.2 mV (Fig. 4Ab). A concurrent decrease in neuronal input resistance was observed in all cells tested, from a mean of 556 ± 28 M at rest to 283 ± 27 M in the presence of muscimol, a 49.6 ± 3.2% decrease and from 584 ± 48 M to 291 ± 36 M, a 48.2 ± 6.9% decrease for the hyperpolarizing and depolarizing response, respectively (Fig. 4B). CACA had no statistically significant effect on either membrane potential or input resistance (n= 4; Fig. 4B).
Application of baclofen induced membrane hyperpolarization in all cells tested, from a mean resting value of -60.0 ± 2.5 mV to -70.1 ± 3.6 mV, a mean peak membrane hyperpolarization of 10.1 ± 1.1 mV (Fig. 5Aa, Ab and B). This hyperpolarization was again associated with a pronounced decrease in neuronal input resistance from a resting value of 398 ± 90 M to 235 ± 82 M, amounting to a 34.4 ± 2.6% decrease (Fig. 5B). All responses persisted in the presence of TTX (500 nM) and were at least partially reversible upon wash.
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Application of bicuculline (10 µM, n= 8) or SR95531 (10 µM, n= 4) induced a significant, reversible reduction in the membrane responses induced by muscimol (Fig. 4Aa–c). Bicuculline application reduced the peak amplitude of muscimol-induced membrane hyperpolarization by 61.7 ± 9.6% from 6.0 ± 0.6 mV to 2.3 ± 1.4 mV in the presence of bicuculline (P < 0.01, Fig. 4B). A 70.4 ± 11.2% (P < 0.001) reduction in the associated input resistance change induced by muscimol was also observed in the presence of bicuculline. Likewise, application of SR95531 reduced the muscimol-induced membrane hyperpolarization by 60.3 ± 6.7%, from 8.8 ± 0.9 mV to 3.5 ± 1.2 mV (P < 0.01, Fig. 4B), accompanied by a 53.5 ± 8.9% (P < 0.03) reduction in the peak input resistance change induced by muscimol.
The effects of GABAB receptor antagonists on agonist-induced responses
Application of 2-OH-saclofen (20 µM, n= 3) or CGP55845(200 nM, n= 5) suppressed membrane responses induced by baclofen (Fig. 5Aa and b). Application of 2-OH-saclofen reversibly reduced the amplitude of membrane hyperpolarization induced by baclofen by 73.1 ± 3.5%, from 10.1 ± 1.1 mV to 2.3 ± 0.3 mV in the presence of this antagonist (P > 0.01, Fig. 5B). A concurrent 45.5 ± 4.4% (P < 0.03) reduction in the peak amplitude of baclofen-induced changes in input resistance was also observed. Bath application of CGP55845reduced the peak amplitude of baclofen-induced hyperpolarization by 86.2 ± 8.4% from 9.8 ± 2.7 mV to 1.8 ± 1.0 mV (P < 0.005, Fig. 5B), with a corresponding 62.3 ± 11.1% reduction in the peak change in input resistance induced by baclofen.
Ionic mechanism underlying GABAA and GABAB receptor agonist-induced hyperpolarization
Similar to evoked IPSPs, with progressive membrane hyperpolarization from rest, the muscimol induced response decreased in amplitude and reversed to a depolarization (Fig. 6Aa) at a membrane potential of -52 ± 9 mV (n= 4, Fig. 6Ab). Current–voltage relationships plotted for four cells, in control conditions and at the peak of the muscimol-induced hyperpolarization (Fig. 6Ba) revealed a reversal potential of -58 ± 4 mV for the muscimol-induced response, again close to the reversal potential for chloride ions under our recording conditions (n= 4, Fig. 6Bb). In voltage clamp at a holding potential of -50 mV, application of baclofen (50 µM, 30 s, n= 5) induced a sustained outward current, with a mean peak amplitude of 52.6 ± 6.5 pA. Voltage ramps from -120 to -60 mV at a rate of 10 mV·s-1 were applied in control conditions and at the peak of the baclofen-induced response to investigate the reversal potential of the baclofen-induced current (Fig. 6Ca). Baclofen-induced outward currents had a mean reversal potential of -95 ± 3.6 mV (n= 5), close to the K+ reversal potential under our recording conditions (Fig. 6Cb)
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When held at membrane potentials close to threshold for firing, in the presence of NBQX (5 µM), D-APV (10 µM), and idazoxan (200 nM) to block any concurrent glutamatergic and noradrenergic synaptic activity, stimulation of the iLF (Fig. 7Aa, n= 6) or cLF (Fig. 7Ab, n= 4) elicited an IPSP which gave rise to rebound firing at the termination of the IPSP. This phenomenon was also observed in some SPNs in response to spontaneous IPSPs (Fig. 7Ac, n= 2 cells). Brief high-frequency stimulation (10–100 Hz) of the iLF or cLF in SPNs under these recording conditions evoked trains of IPSPs which preceded a rebound excitation and bursts of suprathreshold activity (n= 4, Fig. 7Ad). The rebound excitation and the resulting action potential firing were insensitive to strychnine (2 µM), but were completely and reversibly abolished by the subsequent application of bicuculline (10 µM, Fig. 7B).
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The nature of this time-dependent inward rectification was investigated further in a series of current–voltage relationships generated close to threshold (Fig. 8A). The rectification manifests as a slowly developing depolarizing ‘sag’ when hyperpolarizing current pulses generate membrane responses of around -5 to -10 mV (indicated by arrow in Fig. 8A). Application of ZD7288 (10 µM, n= 3), a proposed selective blocker of the hyperpolarization-activated cation current Ih (Harris & Constanti, 1995), was without significant effect on the magnitude of the rectification. However, bath application of Cs+ (1 mM, n= 5) partially reduced the rectification and subsequently higher concentrations of Cs+ (10 mM, n= 4) completely and reversibly blocked the rectification (Fig. 8A and C).
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Discussion |
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Origins of GABA-mediated synaptic inputs
Pharmacological blockade of fast EPSPs revealed fast IPSPs in the majority of SPNs following stimulation of the iLF or cLF, through which descending inputs to SPNs pass. This was observed in both transverse and longitudinal spinal cord slices. IPSPs exhibited a consistent latency, rise time and waveform and no failures in response to repetitive stimulation, indicating that these connections were monosynaptic in origin. The fact that cLF stimulation resulted in IPSPs of monosynaptic origin suggests that pathways originating in the contralateral spinal cord were activated rather than polysynaptic interneuronal circuits within the cord.
The origins of these GABAergic inputs to SPNs are difficult to reconcile in the slice preparation. GABA-mediated IPSPs reported in this study were evoked by stimulation of the iLF and cLF, including SPNs in longitudinal slices, stimulated up to five to six segments away from the recording site. Furthermore, we did not observe any evidence of activation of polysynaptic pathways. These data suggest that the fibres activated are most likely bilateral descending inputs from higher centres and/or intraspinal pathways originating in the lateral funiculi (Jansen & Loewy, 1997). Numerous studies have provided evidence that SPNs receive GABAergic synapses from both supraspinal and intraspinal sources (see Llewellyn-Smith, 2002). A known source of supraspinal GABAergic inputs directly innervating SPNs is the rostral ventrolateral medulla (RVLM; Matsumoto et al. 1994; Miura et al. 1994; Llewellyn-Smith et al. 1995; Deuchars et al. 1997; Stornetta & Guyenet, 1999). Other supraspinal GABAergic neurones which have been shown to functionally or anatomically impinge on SPNs include the gigantocellular depressor area (GiDA) (Aicher et al. 1995; Stornetta & Guyenet, 1999), the rostral ventromedial medulla (parapyramidal area), aspects of the raphe magnus (Loewy, 1981; Millhorn et al. 1987; Sasek et al. 1990) and the nucleus of the solitary tract (NTS; Blessing et al. 1981; Blessing, 1990; Mtui et al. 1993; Lewis & Coote, 1995; Stornetta & Guyenet, 1999). All of these areas have been indicated to project to SPN and use GABA as a neurotransmitter.
Sites of contact of GABA synapses.
Monosynaptic GABA-mediated inputs to SPNs evoked by stimulation of the iLF were relatively fast, and comparable to EPSPs evoked from the same site (Spanswick et al. 1998). The fast rise time is thus consistent with the site of contact being relatively close to the recording site at the soma. Stimulation of the cLF evoked IPSPs with similar but significantly slower rise times suggesting a site of contact at a site relatively distal to those from the iLF, but closer than those evoked by segmental stimulation (see Spanswick et al. 1998). Thus the most likely sites of contact of inputs from both the iLF and cLF are at proximal dendrites or the soma itself. Supporting this notion are spinal transection studies. Descending inputs from these neurones and other higher centres to SPNs descend through the lateral funiculi, from where they branch at right angles towards the IML and SPNs (Anderson et al. 1989; Fuxe et al. 1990). Several days after a complete spinal transection, over 50% of axosomatic GABAergic boutons are lost from SPNs while axodendritic inputs decrease by less than 20% (Llewellyn-Smith & Weaver, 2001). Taken together these data strongly suggest bilateral descending GABAergic inputs to SPNs synapse proximal to the soma. The fact that descending glutamatergic inputs similarly synapse bilaterally, proximal to the soma (Spanswick et al. 1998), suggests a common topographical and functional organization of fast descending inputs to SPNs.
Mechanisms underlying GABA-mediated bilateral synaptic inputs
IPSPs evoked by stimulation of the iLF or cLF were sensitive to bicuculline, generally insensitive to strychnine and reversed polarity close to the reversal potential for chloride ions under our recording conditions. Similarly, IPSPs were mimicked by bath application of GABA and muscimol in the presence of TTX, responses that were also reversibly inhibited by the GABAA receptor antagonists bicuculline and SR95531. Exogenous GABA- and muscimol-induced responses also had reversal potentials indicating chloride selectivity. These data, taken together with the observations that agonists for GABAC receptors were without significant effect on SPN, suggest IPSPs described in this study were mediated by GABAA receptors coupled to a chloride conductance.
It is worthy of note that in a few cells the IPSP was blocked by a combination of strychnine and bicuculline. Despite the prevailing concept that GABA and glycine are two distinct inhibitory transmitter systems, immunocytochemical and ultrastructural studies have demonstrated coexistence of the two amino acids in terminal boutons of some spinal interneurones and colocalization of GABA, glycine, GABAA and strychnine-sensitive glycine receptors at the same synapse (Ornung et al. 1994; Todd et al. 1996; Lévi et al. 1999). Also, a common transporter packages these two inhibitory amino acids into synaptic vesicles (Burger et al. 1991; Chaudhry et al. 1998). Therefore, it has been proposed that GABA and glycine may be released as cotransmitters on postsynaptic targets (Todd et al. 1996). These observations together with the fact that functional studies have shown corelease of the two neurotransmitters from spinal cord interneurones, probably from the same synaptic vesicles (Jonas et al. 1998; O'Brien & Berger, 1999), raises the possibility that GABA and glycine may be coreleased at certain synapses. Whether bicuculline- and strychnine-sensitive IPSPs described in our study are due to the release of GABA and glycine from the same terminal or from different terminals converging onto the same SPN remains to be clarified.
SPNs express GABAB receptors
The selective GABAB receptor agonist baclofen also induced membrane hyperpolarization that was sensitive to the GABAB receptor antagonists 2-hydroxy-saclofen and CGP55845confirming the presence of postsynaptic GABAB receptors in the majority of SPNs. The reversal potential of baclofen-induced responses was close to the reversal potential for K+ ions under our recording conditions, consistent with activation of a potassium-selective conductance. A clear presynaptic role for GABAB receptors has been demonstrated for both SPNs and other central neurones (Dutar & Nicoll, 1988; Bogan et al. 1989; Wu & Dun, 1992; Inokuchi et al. 1992). However, the data described in the present study, to our knowledge, are the first to demonstrate expression of postsynaptic GABAB receptors in SPNs. Despite, the presence of these receptors, indicated by the sensitivity of SPNs to GABAB receptor agonists and antagonists, we were unable to identify a postsynaptic involvement of these receptors in synaptic transmission from either of the lateral funiculi. There are several explanations that may account for our ability to synaptically activate postsynaptic GABAA receptors independent of the GABAB receptors reported here. Firstly, GABAB receptors may be associated with pathways other than those carried by the lateral funiculi, although a presynaptic role for these receptors here has been suggested previously (Wu & Dun, 1992). As already discussed, GABA-expressing interneurones impinge on SPNs. We were unlikely to have recruited these in our study and have not specifically investigated these pathways. However, a presynaptic role for GABAB receptors has also been suggested for inputs evoked by stimulation of the dorsal horn and dorsal roots, presumably involving local spinal circuits and segmental interneurones (Wu & Dun, 1992). Alternatively, GABAB receptors may be localized at extra-synaptic sites and only recruited during prolonged, repetitive stimulation and ‘spill-over’ of GABA into extrasynaptic domains. Such a mechanism is thought to occur at cerebellar (Hamann et al. 2002) and hippocampal (Scanziani, 2000) synapses. However, during the course of our study, repetitive stimulation did not reveal any obvious contribution from GABAB receptors. Further studies are required to clarify the functional role of postsynaptic GABAB receptors in SPNs.
Ih-like time dependent inward rectification
GABA has to date been primarily considered a purely inhibitory transmitter in SPNs, acting to suppress activity (see Coote, 1988). However, thus far, no studies have taken into account how intrinsic membrane properties of SPNs integrate these inputs. Here we have shown that at membrane potentials close to threshold, both spontaneous and evoked IPSPs gave rise to rebound action potential firing and repetitive high frequency (10 Hz) stimulation evoked trains of fIPSPs which summated, followed by a rebound excitation and bursts of action potential firing. These data suggest SPNs have the intrinsic mechanisms to generate auto-rhythmic activity entrained to GABA-mediated synaptic inputs. A current view is that sympathetic rhythmic discharges emerge principally from networks of supraspinal neurones residing within the brainstem (see Barman & Gebber, 2000). This receives support from data that show correlations between brainstem activity and peripherally recorded sympathetic rhythms. Several rhythms within the sympathetic nerve firing patterns have been described including a 0.4–1.2 Hz rhythm referred to as the T-rhythm (Johnson & Gilbey, 1996) and both cardiac and respiratory cycle-related rhythms (McAllen & Malpas, 1997; Malpas, 1998; Barman & Gebber, 2000). Both 10 Hz and 2–6 Hz rhythms have been documented in brainstem nuclei with a number of brainstem sites being suggested as origins of these rhythms, including nuclei containing neurones that use GABA as a neurotransmitter (Barman & Gebber, 1997, 2000). Thus, supraspinal activity may be entrained via a GABA-dependent synaptic mechanism and voltage-dependent conductances expressed by SPNs.
A number of mechanisms have been suggested for the generation of auto-rythmicity in SPNs, including both the presence of low voltage-activated T-type conductances (Wilson et al. 2002), revealed by activation of adrenoceptors (Yoshimura et al. 1987b) and by networks of electrotonically coupled SPNs (Logan et al. 1996). In the present study, we demonstrate a conductance characterized by time-dependent inward rectification observed at membrane potentials close to threshold, activated by hyperpolarization induced by GABAergic IPSPs and mimicked by injection of hyperpolarizing current pulse injection sufficient to drive the membrane potential more negative than around 5–10 mV below threshold. This conductance is reminiscent of the hyperpolarization-activated time-dependent inward rectification (Ih) reported previously in a subpopulation of SPNs in guinea-pig (Inokuchi et al. 1993).
Ih currents are activated by hyperpolarization and have been described in many other neurones and in cardiac myocytes where they are involved in generating pacemaker activity (see Pape, 1996; Luthi & McCormick, 1998; Robinson & Siegelbaum, 2003). The voltage dependence, ion selectivity and kinetics of Ih vary extensively depending on cell-type and thus the properties of Ih are likely to be tailored to its function within the neurone and the circuits in which it resides (Luthi & McCormick, 1998). In relation to this, the Ih-like conductance described here in SPNs differs from those previously reported for other cells in its relative insensitivity to low concentrations of Cs+ and ZD7288, possibly reflecting differences in subunit composition related to SPN-specific functions. Key roles for Ih are the regulation of the response to hyperpolarization resulting from IPSPs and a contribution to pacemaker potentials and rhythm generation (Pape, 1996; McCormick & Bal, 1997; Robinson & Siegelbaum, 2003), for example in thalamocortical relay neurones (McCormick & Bal, 1997) and neurones in the inferior olive (Bal & McCormick, 1997).
In SPN, the Ih-like conductance is a non-inactivating non-selective cation conductance activated at membrane potentials between -50 to -70 mV, with a reversal potential around -30 mV reflecting its permeability to both Na+ and K+ ions. As Ih is activated by hyperpolarization and depolarizes cells towards its reversal potential, it is capable of mediating rebound excitation from periods of inhibition and thus a key property for rhythm generation.
Conclusion
Our results suggest that SPNs receive monosynaptic, bilateral GABAergic inputs, most likely from supraspinal pathways that synapse proximal to the soma to activate postsynaptic GABAA receptors coupled to a chloride conductance. Synaptic activation of a rebound excitation mediated by a time-dependent inward rectification expressed intrinsically by SPNs may provide a novel mechanism enabling SPNs to be entrained to rhythms driven from the brainstem or higher centres.
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) indicating a decrease in input resistance, consistent with opening of ion channels. C, plot of the difference between the steady-state and instantaneous membrane responses to current injection showing ZD7288 (•) had little effect on the inward rectification whereas 1 mM CsCl2 (
) and 10 mM CsCl2 (
), significantly reduced the time-dependent inward rectification compared to control (
).