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. 2007 Aug 9;584(Pt 1):121–136. doi: 10.1113/jphysiol.2007.138198

Altered potassium channel function in the superficial dorsal horn of the spastic mouse

B A Graham 1, A M Brichta 1, P R Schofield 2,3, R J Callister 1
PMCID: PMC2277054  PMID: 17690143

Abstract

The spastic mouse has a naturally occurring glycine receptor (GlyR) mutation that disrupts synaptic input in both motor and sensory pathways. Here we use the spastic mouse to examine how this altered inhibitory drive affects neuronal intrinsic membrane properties and signal processing in the superficial dorsal horn (SDH), where GlyRs contribute to pain processing mechanisms. We first used in vitro patch clamp recording in spinal cord slices (L3–L5 segments) to examine intrinsic membrane properties of SDH neurones in spastic and age-matched wildtype controls (∼P23). Apart from a modest reduction (∼3 mV) in resting membrane potential (RMP), neurones in spastic mice have membrane and action potential (AP) properties identical to wildtype controls. There was, however, a substantial reorganization of AP discharge properties in neurones from spastic mice, with a significant increase (14%) in the proportion of delayed firing neurones. This was accompanied by a change in the voltage sensitivity of rapid A-currents, a possible mechanism for increased delayed firing. To assess the functional consequences of these changes, we made in vivo patch-clamp recordings from SDH neurones in urethane anaesthetized (2.2 g kg−1, i.p.) spastic and wildtype mice (∼P37), and examined responses to innocuous and noxious mechanical stimulation of the hindpaw. Overall, responses recorded in wildtype and spastic mice were similar; however, in spastic mice a small population of spontaneously active neurones (∼10%) exhibited elevated spontaneous discharge frequency and post-pinch discharge rates. Together, these results are consistent with the altered intrinsic membrane properties of SDH neurones observed in vitro having functional consequences for pain processing mechanisms in the spastic mouse in vivo. We propose that alterations in potassium channel function in the spastic mouse compensate, in part, for reduced glycinergic inhibition and thus maintain normal signal processing in the SDH.

The spastic mouse has a pronounced motor phenotype, which is caused by a naturally occurring mutation in the glycine receptor (GlyR). Specifically, intronic insertion of a LINE 1 transposable element in the GlyR β subunit gene causes exon skipping and decreased transcriptional efficiency of the GlyR β subunit (Kingsmore et al. 1994; Mulhardt et al. 1994). The subsequent decrease in GlyR β subunit protein reduces expression of the adult form of the GlyR throughout the nervous system (White & Heller, 1982; Becker, 1990).

As the spastic mouse has an overt motor phenotype work on this mutant has concentrated on central nervous system (CNS) regions related to motor function, such as the ventral horn of the spinal cord (Heller & Hallett, 1982; Biscoe & Duchen, 1986; von Wegerer et al. 2003; Molon et al. 2006) and the hypoglossal nucleus in the brainstem (Callister et al. 1999; Graham et al. 2006). Despite this emphasis, a mutation that reduces GlyRs should impact on function in any CNS region where glycinergic inhibition is important. Consistent with this prediction, we have recently shown that the spastic mutation disrupts inhibition in the superficial dorsal horn (SDH) of the spinal cord (Graham et al. 2003), a sensory region in which glycinergic inhibition contributes to pain processing mechanisms (Zeilhofer, 2005; Lynch & Callister, 2006). In addition to reduced glycinergic inhibition predicted by the spastic mutation, there was a concomitant increase in GABAAergic inhibition. The impact of this altered balance in glycinergic and GABAAergic drive on intrinsic membrane properties has not been investigated. Thus, it remains unclear if normal SDH function is maintained in spastic mice.

A number of studies have now shown that when the balance in synaptic inputs is disrupted in various regions of the nervous system, neurones can compensate by readjusting their intrinsic membrane properties. This form of compensation has been described in the cerebellum (Zhang et al. 2004; Saftenku, 2006), brainstem (Nelson et al. 2003) and hippocampus (van Welie et al. 2004). Thus, one possible consequence of disrupted inhibition in spastic mice may be altered intrinsic membrane properties in SDH neurones.

Neurones in the SDH are a heterogeneous population with four or more different categories described according to AP discharge patterns in response to step-current injection (Thomson et al. 1989; Yoshimura & Jessell, 1989; Lopez-Garcia & King, 1994; Grudt & Perl, 2002; Prescott & De Koninck, 2002; Ruscheweyh & Sandkuhler, 2002; Hu & Gereau, 2003; Lu et al. 2006). Furthermore, studies in the SDH have described conditions where neurones can switch between discharge patterns and thereby adjust their output (Prescott & De Koninck, 2002; Ruscheweyh & Sandkuhler, 2002; Hu & Gereau, 2003). In this study, we compare intrinsic membrane properties and signal processing in SDH neurones from wildtype and spastic mice using both in vitro and in vivo preparations. In vitro patch-clamp recording in transverse spinal cord slices showed that the spastic mutation alters intrinsic membrane properties via changes in the properties of rapid IA potassium currents (IAR). In vivo patch clamping in deeply anaesthetized animals suggested sensory processing mechanisms were not significantly altered by the spastic mutation. Together these in vitro and in vivo data demonstrate clear changes in the behaviour of dorsal horn neurones, which potentially compensate for chronically diminished glycinergic drive in the spastic mouse.

Methods

The University of Newcastle Animal Care and Ethics Committee approved all procedures. Spastic mice (both sexes) backcrossed onto the C57Bl/6 genetic background were obtained from The Jackson Laboratory (Bar Harbour, ME, USA) and bred by mating heterozygous (spa/+) males and females. Using this breeding regime, 25% of the progeny are homozygous and thus exhibit a characteristic motor phenotype. Homozygous-affected spa/spa animals are easily identified approximately 2 weeks after birth according to four criteria: (1) constant resting tremor; (2) clenching of limbs when picked up by the tail; (3) an impaired righting reflex; and (4) a tendency to walk on tip-toes with an arched back (Simon, 1995; Graham et al. 2006). Wildtype C57Bl/6 mice (both sexes) bred and maintained under identical conditions were used as controls. Ages for each genotype are provided in Table 1.

Table 1.

Animal attributes and SDH neurone membrane and AP properties in wildtype and spastic mice from in vitro experiments

Wildtype (n= 97) Spastic (n= 91)
Age (days) 23.3 ± 0.4 22.6 ± 0.5
Weight (g) 10.0 ± 0.3 6.3 ± 0.3 *
Input resistance (MΩ) 523 ± 29 515 ± 29
RMP (mV) −69.2 ± 0.8 −66.6 ± 0.8 *
AP threshold (mV) −39.7 ± 0.8 −38.6 ± 0.6
AP amplitude (mV) 60.4 ± 1.8 61.7 ± 1.8
AP base-width (ms) 3.2 ± 0.1 3.1 ± 0.2
AHP amplitude (mV) −27.8 ± 0.7 −27.6 ± 0.8
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Values are means ±s.e.m.

*

Significant difference between wildtype and spastic indicated (P < 0.05).

In vitro slice preparation

Spinal cord slices were prepared using standard techniques (Yoshimura & Jessell, 1989; Graham et al. 2003). Briefly, animals (wildtype and spa/spa mice) were anaesthetized with ketamine (100 mg kg−1i.p.) and decapitated. Using a ventral approach, the lumbosacral enlargement of the spinal cord was rapidly removed and placed in ice-cold sucrose substituted artificial cerebrospinal fluid (ACSF) containing (mm): 250 sucrose, 25 NaHCO2, 10 glucose, 2.5 KCl, 1 NaH2PO4, 1 MgCl2 and 2.5 CaCl2. Transverse slices (from L3–L5 segments; 300 μm thick) were cut using a vibrating microtome (Leica VT-1000S, Heidelberg, Germany) and then transferred to an incubation chamber containing oxygenated ACSF (118 mm NaCl substituted for sucrose). Slices were allowed to equilibrate for 1 h at room temperature (22–24°C) prior to recording.

In vitro electrophysiology

Slices were transferred to a recording chamber and continually superfused (bath volume 0.4 ml; exchange rate 4–6 bath volumes min−1) with ACSF bubbled with 95% O2 and 5% CO2 to achieve a final pH of 7.3–7.4. All recordings were made at room temperature (22–24°C) from visualized SDH neurones, using near-infrared differential interference contrast optics. Recordings were limited to the SDH (laminae I and II) by targeting neurones located within or dorsal to the substantia gelatinosa (Fig. 1). This region is easily identified by its translucent appearance in transverse spinal cord slices. Patch pipettes (2–4 MΩ) were filled with a potassium methyl sulphate-based internal solution containing (mm): 135 KCH3SO4, 6 NaCl, 2 MgCl2, 10 Hepes, 0.1 EGTA, 2 MgATP, 0.3 NaGTP, pH 7.3 (with KOH). The whole-cell recording configuration was established in voltage-clamp (holding potential −60 mV) and then switched to current-clamp. The membrane potential approximately 15 s after this switch was designated as resting membrane potential (RMP) and all recordings were subsequently made from this potential. All reported membrane potential values have been corrected for a 10 mV junction potential (Barry & Lynch, 1991). In some experiments voltage-clamp recordings were made to examine properties of various subthreshold (i.e. for AP generation) voltage-gated conductances in each genotype. Data were acquired using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) and digitized online (sampled at 10–20 kHz, filtered at 5–10 kHz, respectively) via an ITC-16 computer interface (Instrutech, Long Island, NY, USA) and stored on a Macintosh G4 computer using Axograph v4.6 software (Molecular Devices, Sunnyvale, CA, USA).

Figure 1. Location of SDH neurones recorded in wildtype and spastic mice.

Figure 1

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in vitro A, the location of recorded neurones was plotted on three templates (Paxinos & Watson, 1998) representing L3–L5 spinal segments (see text) that outlined the lamina boundaries of the SDH in the transverse plane (shading shows the dorsal horn template region). B, maps from wildtype mice show recordings were restricted to the SDH (laminae I–II) and sampled the full mediolateral extent of the dorsal horn (left to right). C, maps from spastic mice show recordings were made at similar locations to those from wildtype animals.

The intrinsic membrane properties of SDH neurones were studied by injecting a series of depolarizing and hyperpolarizing rectangular step-currents (800 ms duration, 20 pA increments, delivered every 8 s). During this protocol mean voltage deflections (i.e. in parts of the voltage trace not containing AP discharge) were limited to −20 mV during depolarizing steps, and −100 mV during hyperpolarizing steps.

To activate and identify ionic currents of interest, an initial voltage-clamp protocol was applied (holding potential, −60 mV). This protocol delivered a hyperpolarizing step to −90 mV (1 s duration) followed by a depolarizing step to −40 mV (200 ms duration). By limiting depolarization to −40 mV we avoided activation of tetrodotoxin-sensitive Na+ and delayed rectifier channels (Safronov, 1999). The protocol used the automated P/N leak subtraction method, within the Axograph software, to remove both capacitive and leakage currents (Sontheimer & Ransom, 2002). This protocol identified the four major ionic currents previously described in SDH neurones, including the outward potassium currents (rapid and slow IA) and the inward currents, T-type calcium and non-specific cationic Ih (Yoshimura & Jessell, 1989; Grudt & Perl, 2002; Ruscheweyh & Sandkuhler, 2002; Ruscheweyh et al. 2004).

For neurones with rapid IA currents (IAr) an additional protocol was used that characterized voltage-dependent activation and steady-state inactivation. Voltage-dependent activation was assessed by applying a hyperpolarizing prepulse to −90 mV (1 s duration) followed by depolarizing voltage steps of increasing amplitude (−85 mV to −40 mV, 5 mV increments, 200 ms duration). Steady-state inactivation was assessed by applying hyperpolarizing prepulses of decreasing amplitude (−90 mV to −40 mV, 5 mV increments, 1 s duration), followed by a depolarizing voltage step to −40 mV (200 ms duration).

Neurone location in SDH slices

At the completion of in vitro recording for a given neurone, the dorsal horn was photographed using an Olympus DP50 digital camera and Viewfinder lite software (Olympus, Tokyo, Japan), while the patch pipette was still attached to the neurone in situ. Subsequently, three drawings (adapted from Paxinos & Watson, 1998) of the dorsal horn in the transverse plane (segments L3, L4, L5), outlining the grey and white matter borders, were used as templates to plot each neurone's position. Dorsal horn images were matched to one of these templates according to: (1) the extent of the dorsal columns in the mediolateral axis; (2) the size and shape of the dorsal grey matter; and (3) the distance from the central canal to the apex of the dorsal columns. In L3 slices, the dorsal columns cover the largest area in the mediolateral axis, the SDH is relatively small, and the distance between the central canal and the dorsal columns is minimal. L4 slices have dorsal columns that extend less in the mediolateral axis, have a broad SDH that extends laterally, and the distance between the central canal and the dorsal columns is minimal. L5 slices have relatively small dorsal columns, a broad SDH, and the distance between the central canal and dorsal columns is maximal. The appropriate template for the photographed slice was superimposed and rescaled in the dorsoventral and mediolateral axes to optimize its fit to the SDH image. Patch pipette tip position was then plotted on the selected template. All templates were then rescaled back to their original dimensions with recording location preserved. Finally, templates were grouped and overlayed, collapsing all recording locations in slices from each spinal segment (see Fig. 1).

Data analysis

Criteria for inclusion of a neurone in the in vitro data set was a RMP more negative than −50 mV and a series resistance < 20 MΩ (filtered at 5 kHz). All data were analysed offline using Axograph software. Individual APs elicited by step-current injection were captured using a derivative threshold method (dV/dt= 15 V s−1) with the inflection point during spike initiation being defined as AP threshold. Rheobase current was defined as the smallest step-current to elicit at least one AP. The difference between AP threshold and its maximum positive peak was defined as AP amplitude. AP base-width was measured at AP threshold. AP afterhyperpolarization (AHP) amplitude was measured as the difference between AP threshold and the maximum negative peak following the AP. Two parameters were used to classify each neurone's AP discharge pattern during step-currents: discharge latency was taken as the time from the onset of step-current injection to the first evoked AP; and discharge duration was defined as the time between the onset of the first and last AP during a step-current.

Features of IAr, the dominant current in SDH neurones (Ruscheweyh & Sandkuhler, 2002), were analysed in detail. IAr was activated by applying a voltage step protocol (−90 mV to −40 mV), which maintained membrane potential below activation threshold of tetrodotoxin (TTX)-sensitive sodium channels. TTX was not used in these experiments because it would have precluded study of AP discharge properties in the same neurone. We repeated the above protocol four times to obtain an average response for analysis. IAr amplitude was measured by subtracting the amplitude of any steady-state potassium current component (in the last 50 ms of the step to −40 mV) from the initial IAr current peak (Ruscheweyh & Sandkuhler, 2002). The decay phase of the IAr response was fitted with a single exponential (over 20–80% of the falling phase). IAr activation and steady-state inactivation were analysed by normalizing all current amplitudes to the response during the −90 mV to −40 mV step (maximal IAr activation in our protocol). Data were fitted with the Boltzmann equation: g/gmax= 1/[1 + exp(V−VH)/k], where g/gmax is normalized conductance, V is membrane potential, VH is voltage at half-maximal activation (or inactivation), and k is the slope factor. Most drugs used to study voltage-gated ionic currents were obtained from Sigma Aldrich (Sydney, Australia). In some experiments TTX was applied to prevent epileptiform activity (see Fig. 4) and was obtained from Alomone Laboratories (Jerusalem, Israel).

Figure 4. Pharmacological identification of voltage-activated currents in SDH neurones.

Figure 4

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A–D, all currents were recorded during a voltage-clamp protocol (bottom trace in D) that stepped membrane potential from −60 mV to −90 mV (1 s duration), then to −40 mV (200 ms), and returning to −60 mV. Currents recorded in: control solutions are red; in the presence of specific blockers are blue; and after washout (10 mins) are orange. A and B also show current traces recorded in low blocker concentrations (green). A, outward currents featuring rapid activating and inactivating kinetics at the onset of depolarization (−40 mV step) were relatively insensitive to low concentration of 4AP (0.5 mm), but were completely abolished by high concentration of 4AP (5 mm) (see inset). This kinetic and pharmacological profile confirms their identity as rapid A-currents (IAr). B, outward currents that activated less rapidly and inactivated slowly during depolarization (−40 mV step) were highly sensitive to both low (0.5 mm) and high (5 mm) concentrations of 4AP. This analysis confirms their identity as slow A-currents (IAs). C, inward currents featuring rapid activating and inactivating kinetics at the onset of depolarization (−40 mV step) were Ni2+ (100 μm) sensitive. This profile confirms their identity as T-type calcium currents. D, slowly activating inward currents observed during hyperpolarization (−90 mV step) were Cs+ (2 mm) sensitive, confirming their identity as Ih current. Note, TTX (1 μm) was present in the bathing solution for all experiments where 4AP was applied to prevent epileptiform activity previously described in SDH neurones (Ruscheweyh & Sandkuhler, 2003).

In vivo surgery and electrophysiology

Details of the in vivo mouse spinal cord preparation have been previously described (Graham et al. 2004a,b). Briefly, animals (C57Bl/6 mice, both sexes, aged 21–66 days, see Table 2) were anaesthetized with urethane (2.2 g kg−1i.p.). A laminectomy exposed the lumbosacral enlargement and recordings were obtained from SDH neurones (L4–L5 segments), using an AxoClamp 2B amplifier (Molecular Devices, Sunnyvale, CA, USA). Patch pipettes (8–12 MΩ) were filled with the same KCH3SO4 internal solution as used for in vitro recordings. The exposed surface of the spinal cord was continually irrigated with warmed (37°C) ACSF. Data were digitized online as described in the in vitro experiments above. After obtaining the whole-cell recording configuration in voltage-clamp (series resistance < 30 MΩ), the recording mode was switched to current-clamp. The membrane potential approximately 15 s after this switch was designated as RMP and all recordings were subsequently made from this potential. Membrane potentials were corrected for a 10 mV junction potential (Barry & Lynch, 1991). At the completion of experiments animals were killed with an overdose of Nembutal (100 mg kg−1i.p.).

Table 2.

Animal attributes and SDH neurone membrane properties in wildtype and spastic mice from in vivo experiments

Wildtype (n= 37) Spastic (n= 32)
Age (days) 37.3 ± 0.8 36.4 ± 2.1
Weight (g) 15.4 ± 0.3 12.5 ± 0.8*
Recording depth† (μm) 239 ± 9 214 ± 11
Input resistance (MΩ) 399 ± 37 334 ± 25
RMP (mV) −57.9 ± 0.9 −58.6 ± 1.0
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†

Recording depth denotes distance of recorded neurone from dorsal surface of spinal cord. Means ±s.e.m. with number of neurones in parentheses.

*

Significant difference between wildtype and spastic indicated (P < 0.05).

Classification of in vivo responses

The receptive field of each neurone was first located by stroking a soft-bristled brush over the glabrous skin of the animal's hindpaw. The centre of the receptive field was then stimulated: first with the brush (approx. 1 s) and then, by pinching (1 s pinch ∼100 g mm−2) with a custom-built computer-controlled pincher (Graham et al. 2004a). SDH neurones were functionally classified by evaluating brush and pinch responses (Graham et al. 2004b). Only neurones where responses to both brush and pinch stimuli could be characterized were included in this analysis. Neurones were classified as nociceptive if they responded maximally to noxious ‘pinch’ stimulation. Neurones were classed as light touch if they responded maximally to innocuous brush. Neurones that did not discharge APs during hindpaw stimulation were classified into two groups. Subthreshold neurones responded to brush and pinch with depolarizations but failed to discharge APs. Hyperpolarizing neurones responded to application of the test stimuli with membrane hyperpolarization, which was most apparent during pinch.

Statistics

Statistical analysis was carried out using SPSS v10 (SPSS Inc., Chicago, IL, USA). Student t tests were used to compare variables between genotypes. Data that failed Levene's test for homogeneity of variance were compared using the non-parametric Kruskal–Wallace test. G tests, with Williams' correction, were used to determine if discharge patterns, hyperpolarizing responses, voltage-activated currents or functional responses differed between genotypes (Sokal & Rohlf, 1981). G tests, were also used to compare relationships between discharge patterns, hyperpolarizing responses, voltage-activated currents within wildtype and within spastic neurones. Correspondence analysis, with canonical (symmetrical) normalization, was then used to identify where the strongest relationships occurred in the above G test comparisons. Statistical significance was set at P < 0.05. All values are presented as means ±s.e.m.

Results

In vitro patch-clamp recordings were obtained from 208 SDH neurones in wildtype and spastic mice (117 and 91 neurones from 20 and 14 animals, respectively). These experiments tested whether previously described disruptions to fast synaptic inhibition in spastic mice (Graham et al. 2003) influenced the intrinsic membrane properties of SDH neurones. The results of in vivo patch-clamp recordings from 69 SDH neurones in wildtype and spastic mice are also presented (37 and 32 neurones from 22 and 20 animals, respectively). These experiments assessed whether processing of innocuous and noxious sensory signals were altered in the SDH of spastic mice.

Intrinsic membrane properties of SDH neurones recorded in vitro

The location of neurones recorded in wildtype and spastic animals is shown in Fig. 1 and indicates that similar regions of the SDH between L3 and L5 spinal levels were sampled in both genotypes. Animal attributes, neurone membrane and AP properties recorded in wildtype and spastic mice are presented in Table 1. Although age-matched, spastic animals were significantly smaller (6.3 ± 0.3 g) than their wildtype counterparts (10.0 ± 0.3 g). Apart from a modest but significant depolarization of RMP (spastic−66.6 ± 0.8 mV versus wildtype −69.2 ± 0.8 mV), SDH neurones in the spastic mouse have membrane and AP properties that are identical to those of wildtype animals.

We next compared SDH neurone responses to depolarizing step-current injections. The responses (hereafter termed AP discharge patterns) observed in wildtype and spastic mice are summarized in Fig. 2 and are qualitatively similar to those reported in previous organotypic culture (Lu et al. 2006), acute dissociation (Hu & Gereau, 2003), in vitro (Thomson et al. 1989; Yoshimura & Jessell, 1989; Lopez-Garcia & King, 1994; Prescott & De Koninck, 2002; Ruscheweyh & Sandkuhler, 2002; Balasubramanyan et al. 2006) and in vivo (Graham et al. 2004b) studies. Based on AP discharge pattern, neurones fell into one of four main groups: Tonic firing neurones, exhibit sustained repetitive AP discharge during multiple step-current injections; Initial bursting neurones, exhibit brief repetitive AP discharge during multiple step-current injections where APs are confined to step-current onset; Delayed firing neurones, respond only at higher intensity step-current injections and exhibit substantial delay between current onset and AP discharge; and Single spiking neurones, respond only at high intensity step-current injections and discharge no more than one or two APs at current onset. All four discharge patterns were observed in both genotypes; however, the proportion of neurones exhibiting each discharge pattern differed (Fig. 2, right bar plots). The incidence of initial bursting and single spiking discharge patterns was reduced in spastic mice (37% to 24%; 21% to 13%, respectively), whereas the proportion of tonic firing and delayed firing discharge patterns was increased (13% to 21%; 29% to 42%, respectively). These results indicate that the spastic mutation causes substantial reorganization of AP discharge properties in SDH neurones.

Figure 2. Depolarization-activated responses in wildtype and spastic mice recorded in vitro.

Figure 2

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A, SDH neurones in wildtype exhibited four distinct AP discharge patterns during depolarizing current-step injections (responses shown to steps of 40, 80 and 120 pA): tonic firing, initial bursting, delayed firing and single spiking (see text for details). In wildtype mice initial bursting was the most common discharge pattern, followed by delayed firing, single spiking and tonic firing. B, SDH neurones in spastic mice also exhibited tonic firing, initial bursting, delayed firing and single spiking during depolarizing current-step injections (responses shown to steps of 40, 80 and 120 pA). The incidence of each discharge pattern in spastic mice, however, differed from that in wildtype mice. In spastic mice, the proportion of neurones exhibiting delayed firing and tonic firing increased, while those showing initial bursting and single spiking decreased. Distribution of discharge patterns for each genotype were compared using 2 × 4 G tests for goodness of fit and were found to be significantly different (G statistic = 7.895, P < 0.05).

SDH neurones also exhibit distinctive responses to hyperpolarizing step-currents (Yoshimura & Jessell, 1989; Lopez-Garcia & King, 1994). Therefore we characterized these responses in wildtype and spastic mice (Fig. 3). As with discharge patterns, SDH neurones in both genotypes exhibited a range of responses as previously described in vitro (Yoshimura & Jessell, 1989; Lopez-Garcia & King, 1994) and in vivo (Graham et al. 2004b). Most neurones (57% and 58% for wildtype and spastic, respectively) exhibited passive responses, during and after the hyperpolarizing step-current injection, with membrane potential returning to RMP without apparent active components (Fig. 3A and B, bottom traces). Some neurones (∼25%) exhibited rebound depolarization at the cessation of step-current injection, which often elicited AP discharge (15/24 in wildtype and 17/22 in spastic; Fig. 3A and B, top traces). In both genotypes, about 20% of neurones exhibited a long-lasting hyperpolarization during large-amplitude hyperpolarizing step-current injections that continued after the cessation of step-current injections (Fig. 3A and B, middle trace). Unlike the responses to depolarizing current injection (Fig. 2) the proportions of the three hyperpolarizing responses were similar in wildtype and spastic mice (Fig. 3, right bar plots). These results suggest that responses to hyperpolarization are unaffected by the spastic mutation.

Figure 3. Hyperpolarization-activated responses in wildtype and spastic mice recorded in vitro.

Figure 3

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A, SDH neurones in wildtype mice exhibited three types of response during hyperpolarizing current-step injections (responses shown to steps of −20, −40, −60, −80 and −100 pA): rebound, hyperpolarizing and passive (see text for details). In wildtype mice passive responses were observed in 60% of neurones, and the remaining 40% was split evenly between rebound and hyperpolarizing responses. B, SDH neurones in spastic mice also exhibited rebound, hyperpolarizing and passive responses during hyperpolarizing current-step injections (responses shown to steps of −20, −40, −60, −80 and −100 pA). The proportion of SDH neurones exhibiting the three responses in spastic mice was similar to wildtype mice (G statistic = 0.146, P= 0.95). The unshaded portion of the columns for rebound neurones (R) denotes neurones where AP discharge was absent.

It is well established that the range of depolarizing and hyperpolarizing responses observed in SDH neurones is caused by differences in the expression of a number of voltage-gated ion channels (Yoshimura & Jessell, 1989; Ruscheweyh & Sandkuhler, 2002; Melnick et al. 2004a,b; Ruscheweyh et al. 2004). Thus, to evaluate the effect of the spastic mutation on voltage-gated ion channel expression we applied a voltage-step protocol that permitted identification of four major voltage-gated currents previously described in these neurones (Yoshimura & Jessell, 1989; Grudt & Perl, 2002; Ruscheweyh & Sandkuhler, 2002). We first characterized these voltage-activated currents pharmacologically in wildtype mice to confirm that they could be reliably identified according to their direction (inward or outward) and kinetic features (Fig. 4).

Two outward currents with features of A-type potassium currents (activated by stepping from −90 mV to −40 mV) were identified (Yoshimura & Jessell, 1989; Ruscheweyh et al. 2004). One had rapid activation and inactivation kinetics (rapid A-current; IAr) and the other activated more slowly and was not completely inactivated by the end of the depolarizing step (slow A-current; IAs). These currents could also be identified by their differing sensitivity to 4-aminopyridine (4AP; Fig. 4A versus B). IAr was relatively insensitive to 0.5 mm 4AP (39% block, n= 8), whereas IAs was blocked by this concentration (n= 4). In contrast, both forms of IA were completely blocked by 5 mm 4AP (n= 8 and n= 4, respectively).

Two inward currents were also identified. Some neurones expressed an inward current during depolarization (−90 mV to −40 mV). These currents had rapid activation and inactivation kinetics and were sensitive to Ni2+ (100 μm; 70% block, n= 4, Fig. 4C). These features are consistent with those of low threshold activated ‘T-type’ calcium currents (Yoshimura & Jessell, 1989). Other neurones expressed a slowly activating inward current during hyperpolarization (−60 mV to −90 mV) that was sensitive to Cs+ (2 mm; 94% block, n= 4. Fig. 4D). The characteristics of this current are consistent with those of the non-selective cation current Ih (Yoshimura & Jessell, 1989; Grudt & Perl, 2002; Ruscheweyh & Sandkuhler, 2002). Finally, no response to the above protocol could be resolved in some neurones from background membrane noise (∼10% in both genotypes). Currents of interest in these neurones were either absent or below detection level. We cannot rule out their presence as the current source may have been electrotonically distant from the recording electrode (Lorincz et al. 2002; Rateau & Ropert, 2006).

The above experiments confirmed our ability to differentiate four main voltage-activated currents in SDH neurones according to current direction and kinetics. Perhaps surprisingly, our studies indicate that in most cases (19/20 neurones) one of these four currents was dominant. Only one neurone in our sample displayed multiple currents (ICa and Ih). We acknowledge, however, that multiple currents may exist but fall below detection levels. Figure 5 shows examples of the four current species recorded in wildtype and spatic mice. These currents were expressed in both genotypes and appeared qualitatively similar in terms of amplitude and kinetics. Bar plots in Fig. 5 (right) summarize the distribution of these currents in wildtype and spastic genotypes. IAr was present in 40% of recordings, while IAs, ICa, and Ih each constituted 10–20% of the samples. Surprisingly, there were no significant differences in the distribution of these voltage-gated currents on SDH neurones in the two genotypes despite substantial reorganization of AP discharge patterns in the spastic mouse (Fig. 2).

Figure 5. Voltage-activated currents in SDH neurones from wildtype and spastic mice recorded in vitro.

Figure 5

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A, representative voltage-clamp traces showing voltage-activated currents recorded in SDH neurones from wildtype mice during a voltage step protocol (shown below current traces in A and B). Four different currents were readily identified: a rapid outward current with features consistent of IAr; a slower outward current that had characteristics of a IAs; a rapid inward current with features consistent with a T-type ICa; and, a slow hyperpolarization-activated current with characteristics of the nonselective cation current Ih. The predominant current was IAr in recordings from wildtype mice (> 40%). The remaining three currents (IAs, T-type ICa, and Ih) made up 10–20% each of the sample. B, representative voltage-clamp traces showing examples of IAr, IAs, ICa and Ih recorded in SDH neurones from spastic mice. The proportion of SDH neurones expressing each voltage-activated current in spastic mice was similar to that in wildtype mice (G statistic = 0.971, P= 0.91). Note, in both genotypes some neurones (10–20%) exhibited no prominent voltage-activated currents during the step protocol and were therefore not classified (NC).

To examine the relationship between specific discharge patterns, hyperpolarizing responses and voltage-activated currents we analysed data from a sample of neurones where all three properties were obtained (wildtype n= 35; spastic n= 47). G tests were used to identify if significant relationships existed and then a series of two-dimensional Correspondence analysis maps were generated to resolve specific associations between: discharge pattern and hyperpolarizing response (Fig. 6A); discharge pattern and voltage-activated current (Fig. 6B); and, hyperpolarizing response and voltage-activated current (Fig. 6C). These maps transform row and column data from contingency tables and depict them in two-dimensional space. This provides a graphical representation of relationships between variables where proximity indicates relationship strength.

Figure 6. Correspondence analyses of discharge patterns, hyperpolarizing responses and voltage-activated currents in wildtype and spastic mice.

Figure 6

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A–C, correspondence maps were generated from contingency tables comparing: A, discharge patterns and hyperpolarizing responses; B, discharge patterns and voltage-activated currents; and C, hyperpolarizing responses and voltage-activated currents. In these plots the strength of relationships between variables is indicated by the proximity of data points. Data are transformed into an ‘equivalent’ space where the largest proportion of variability in the data points is captured in the first dimension and the next largest in the second dimension. For each plot the proportion of the variability captured by each axis is shown in parentheses. The primary variables are plotted with surrounding territories (filled circles with shaded clouds), such that each level is 0.25 of a dimensional unit (arbitrarily chosen to assist identification of associations). The relationships of comparison variables (open circles) are then determined by identifying in which primary variable territories they appear. A, discharge patterns and hyperpolarizing responses were highly correlated in wildtype and spastic mice (G statistic = 28.26 and 32.291, d.f. = 6, respectively, both P < 0.001). In wildtype mice the strongest relationships were between initial bursting and rebound responses, and delayed firing-single spiking and passive responses. Similarly, in spastic mice, delayed firing and single spiking and passive responses were also related but in addition tonic firing and rebound response showed a strong relationship. B, discharge patterns and voltage-activated currents were uncorrelated in wildtype mice but highly correlated in spastic mice (G statistic = 16.513 and 45.871, respectively, d.f. = 12, P= 0.2 and < 0.001). The major difference in the two genotypes was a strengthening of the relationships between delayed firing and IAr, and tonic firing and ICa–Ih. C, hyperpolarizing responses and voltage-activated currents were correlated in wildtype and spastic mice (G statistic = 25.327 and 58.490, d.f. = 8, respectively, both P < 0.001). Strong relationships between passive and IAr–NC, rebound and ICa–Ih, and hyperpolarizing–IAs were evident in both genotypes.

The above analysis revealed several potentially important relationships. First, the relationship between discharge patterns (tonic firing, initial bursting, delayed firing and single spiking) and hyperpolarizing responses (rebound, hyperpolarizing and passive) was significant in neurones from both genotypes. Delayed firing and single spiking were associated with passive responses in both genotypes whereas initial bursting or tonic firing was associated with rebound responses in wildtype and spastic neurones, respectively. Second, the relationship between discharge patterns and voltage-activated currents (IAr, IAs, ICa and Ih) was significant in spastic neurones but not wildtype. For example, in spastic neurones there was an association of tonic firing with ICa and Ih, and delayed firing with IAr. Third, the relationship between hyperpolarizing responses and voltage-activated currents was significant in neurones from both spastic and wildtype. For example, associations existed between hyperpolarizing responses and IAs, rebound responses and ICa/Ih, and passive responses with IAr or an absence of all four currents.

Two lines of evidence suggest a role for IAr may underlie the greater prevalence of delayed firing in spastic animals (Fig. 2B). First, our Correspondence analysis showed an increase in the association between delayed firing and IAr in spastic mice. Second, pharmacological experiments have shown previously that blocking IAr can prevent delayed firing (Yoshimura & Jessell, 1989; Ruscheweyh & Sandkuhler, 2002; Ruscheweyh et al. 2004). Therefore, we studied the kinetic properties of IAr in wildtype and spastic mice (Fig. 7). Comparisons of maximum peak current (250 ± 27 pA versus 257 ± 26 pA), rise time (7.5 ± 0.5 ms versus 8.6 ± 1.5 ms, respectively) and decay time constant (47.6 ± 4.0 ms versus 56.1 ± 7.6 ms, respectively) indicate that these IAr properties were similar in wildtype and spastic mice (Fig. 7B). Figure 7C compares the voltage sensitivity of activation and steady-state inactivation of IAr for the two genotypes. The voltage sensitivity of IAr activation was similar in wildtype and spastic mice. In contrast, steady-state inactivation of IAr is shifted to the right in spastic animals at membrane potentials between −90 mV and −70 mV. This suggests that IAr is more capable of delaying AP discharge in spastic mice and provides a possible mechanism for increased delayed firing (Fig. 2B) in the SDH of mice carrying the spastic mutation.

Figure 7. Comparison of IAr properties in wildtype and spastic mice recorded in vitro.

Figure 7

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A, representative voltage-clamp recordings of IAr in wildtype and spastic mice during a voltage step protocol (shown below each trace). Bar graphs compare peak amplitude, rise time and decay time constant for IAr in wildtype and spastic mice. B, representative recordings of IAr showing steady-state inactivation in wildtype and spastic mice during a voltage step protocol (shown below traces). In spastic mice larger responses are clustered near maximum amplitude. In wildtype recordings responses are spread more evenly. In contrast, IAr activation was similar in both genotypes (not shown). C shows steady-state inactivation (circles) and activation curves (squares) constructed with group data. There is a depolarizing shift in the steady-state inactivation of IAr in spastic mice, whereas activation curves are similar for both genotypes.

Processing of innocuous and noxious signals in vivo

To study the functional consequences of our in vitro findings in spastic mice we made in vivo patch-clamp recordings from SDH neurones and examined responses to innocuous and noxious mechanical stimulation of the hindpaw. Data from spastic animals were compared with those previously reported for age-matched wildtype mice (37.3 ± 0.8 days versus 36.4 ± 2.1 days) (Graham et al. 2004b). Table 2 presents the membrane properties of in vivo recorded neurones. Input resistances and RMPs were similar for both genotypes (399 ± 37 MΩversus 334 ± 25 MΩ, and −57.9 ± 0.9 mV versus−58.6 ± 1.0 mV, respectively). Together, these data suggest that the spastic mutation does not alter passive membrane properties of SDH neurones in vivo. In terms of functional activation, similar responses to noxious an innocuous stimulation were recorded in spastic SDH neurones to those previously reported in wildtype (Graham et al. 2004b). Four response classes, based on AP discharge and subthreshold membrane response were observed (nociceptive, light touch, subthreshold and hyperpolarizing; Fig. 8). The incidence of each response class did not differ in wildtype and spastic mice (P= 0.21). This analysis suggests that processing mechanisms in the SDH are preserved in the spastic animals.

Figure 8. Functional responses of wildtype and spastic SDH neurones recorded in vivo.

Figure 8

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A, representative responses recorded in wildtype SDH neurones during innocuous (brush) and noxious (pinch) stimulation of the hindpaw. Responses were classified into four groups (see text for details): nociceptive, light touch, subthreshold and hyperpolarizing. Nociceptive responses account for > 50% of the sample while subthreshold and hyperpolarizing responses are rare (∼10% each). B, representative nociceptive, light touch, subthreshold and hyperpolarizing responses recorded in spastic SDH neurones. The proportion for each functional class in this sample is similar in wildtype and spastic animals (G statistic = 3.23, P= 0.21). Since there is no shift in the distribution of functional responses this suggests sensory processing is preserved in the SDH of spastic mice. Note that data for wildtype animals has been presented previously (Graham et al. 2004b).

Altered spontaneous background discharge

The wildtype data set presented in our previous study (Graham et al. 2004a) contained a small minority of neurones (11%, 4/37) that exhibited low-frequency (< 1 Hz) spontaneous discharge that was unrelated to stimulation (Fig. 9A, upper trace). A population of spontaneously active neurones was also present in recordings made from spastic SDH neurones (12%, 4/32), but with significantly higher discharge frequency (8.4 ± 3.2 Hz versus < 1 Hz, P < 0.05). In three of these four spastic neurones hyperpolarizing responses during pinch temporarily blocked discharge in these spontaneously active neurones (Fig. 9B). Interestingly, post-pinch discharge frequency was significantly elevated above pre-pinch levels in all spontaneously active spastic neurones. Comparison of mean AP frequency (recorded 2 s pre-pinch) with mean AP frequency (2 s post-pinch) indicates a 185 ± 31% increase in discharge rate. These data indicate that a small population of SDH neurones in spastic mice behave in a manner contrary to the overall trend towards decreased activity as revealed in our in vitro recordings.

Figure 9. Spontaneous AP discharge frequency is increased in a subpopulation of spastic SDH neurones recorded in vivo.

Figure 9

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A, spontaneous AP discharge unrelated to hindpaw stimulation was rare (4/37 neurones) and of low frequency (< 1 Hz) in wildtype mice (upper trace). A similar low proportion of SDH neurones exhibited spontaneous AP discharge (4/32 neurones) in spastic mice. Discharge rate in these recordings, however, was significantly higher (8.4 ± 3.2 Hz, lower trace). B, most spontaneously active SDH neurones in spastic mice (3/4) exhibited hyperpolarizing responses during hindpaw pinch and elevated post-pinch discharge. Similar post-pinch responses were not observed in recordings from wildtype mice.

Discussion

By combining in vitro and in vivo experiments we have shown that defects in the inhibitory GlyR result in changes to neuronal intrinsic membrane properties that appear to influence sensory processing mechanisms in the SDH of the spastic mouse. These findings indicate that in addition to the overt motor symptoms of the spastic phenotype, dysfunction also exists in an important sensory region of the spinal cord where GlyRs contribute to pain processing.

Reorganization of AP discharge properties in the spastic mouse

Between four and six distinct forms of AP discharge are typically identified in SDH neurones in vitro (Thomson et al. 1989; Yoshimura & Jessell, 1989; Lopez-Garcia & King, 1994; Grudt & Perl, 2002; Prescott & De Koninck, 2002; Ruscheweyh & Sandkuhler, 2002; Balasubramanyan et al. 2006). In agreement with these studies, we describe four major AP discharge patterns (tonic firing, initial bursting, delayed firing and single spiking) in the SDH of wildtype and spastic mice. Each discharge pattern is present in both genotypes; however, there is a shift in the incidence of each category in spastic mice, the most prominent being an increase in delayed firing neurones (Fig. 2B).

Our search for mechanisms that would explain this increase in delayed firing neurones revealed a shift in IAr steady-state inactivation in spastic mice (Fig. 6C). This would allow IAr to restrain AP discharge and effectively increase the incidence of delayed firing in spastic SDH neurones. Such specific and selective alterations to the voltage dependence of IA steady-state inactivation has been observed in other studies. For example, in hypothalamic and hippocampal neurones the voltage dependence of IA steady-state inactivation is shifted to more depolarized potentials (as observed in our study) by cannabinoids, acting through the CB1 receptor (Deadwyler et al. 1995; Tang et al. 2005). This raises the intriguing possibility that the endocannbinoid system may be more engaged in spastic mice. Alternatively, two species of IA currents identified in this study (IAr and IAs) have been shown to differ in voltage dependence of steady-state inactivation and so a change in the relative expression of these two currents may explain our result (Ruscheweyh et al. 2004). This is unlikely, however, because we could easily differentiate between IAr and IAs (Fig. 5) and we did not detect any change in the decay of IA currents (used to differentiate IAr and IAs). Future experiments that manipulate the endo-cannabinoid system and characterize potassium channel subunit expression in wildtype versus spastic mice will be required to directly test these hypotheses.

Despite our focus on mechanisms that might increase the incidence of delayed firing, it is clear that additional processes occur in spastic mice to alter the incidence of the other discharge categories: i.e. increasing tonic firing and decreasing initial bursting and single spiking. A relationship between these other discharge patterns has been described (Melnick et al. 2004a), suggesting that the expression levels of voltage-gated Na+ channels and delayed rectifier K+ channels determine these discharge patterns. Thus, disturbed expression of these channels in spastic SDH neurones may alter the incidence of discharge patterns. Regardless of how this redistribution of discharge patterns occurs, we have recently shown that tonic firing and initial bursting neurones may have similar roles in nociceptive processing under in vivo conditions (Graham et al. 2007a).

Functional responses in the spastic mouse

The data presented above and in our other in vitro investigation of the spastic mutation (Graham et al. 2003) indicate that various properties of SDH neurones are altered. The in vitro data, however, cannot address how such changes influence sensory processing of peripheral stimuli. To answer this question we made in vivo patch-clamp recordings from SDH neurones in spastic mice. Our data suggest that sensory processing is not markedly altered in the SDH of the spastic mouse (Fig. 8B). There was, however, a small population of neurones in spastic animals with elevated rates of spontaneous discharge as well as post-pinch discharge (Fig. 9). The existence of changes that don't apply to every SDH neurone is consistent with the well-documented heterogeneity and complex circuitry in the SDH (Grudt & Perl, 2002; Ruscheweyh & Sandkuhler, 2002; Lu & Perl, 2003, 2005; Graham et al. 2007b).

The results of our in vitro experiments, here and those reported previously (Graham et al. 2003), may provide explanations for the apparent preservation of normal signal processing in the SDH of spastic mice recorded in vivo. First, the enhanced capacity of IA to inhibit depolarization-evoked AP discharge in spastic SDH neurones is one mechanism that would supplement normal inhibitory mechanisms in the spastic mouse where glycinergic inhibition is decreased. It is also possible that the increase in GABAAergic inhibition we have previously described in spastic mice provides additional synaptic inhibition and dampens SDH neurone responses. GABAAergic currents have much slower decay kinetics than glycinergic currents (mean decay time constants of 10.5 ± 0.6 ms versus 27.4 ± 1.8 ms; Graham et al. 2003) in the SDH and their increased expression in spastic mouse may actually provide an increased level of inhibition. Finally, the impact of IA could be enhanced by hyperpolarization provided by increased GABAAergic inhibition. Our in vivo data do not allow us to differentiate between these proposed mechanisms (i.e. compensation by rapid A potassium currents, increased GABAAergic currents, or a combination of both).

The increased spontaneous background activity in some SDH neurones from spastic mice, in vivo, is less easily explained by our in vitro data. Spontaneously active neurones constituted only a small proportion of our in vivo sample (wildtype 11%, spastic 12%) but we did not observe spontaneous activity in vitro. One explanation for this discrepancy is that the membrane potential of SDH neurones in vivo was approximately 10 mV more depolarized than in vitro. Thus, the hyperpolarized state of in vitro neurones may have prevented spontaneous activity in some wildtype and spastic neurones. Alternatively, the vast difference in the preserved neuronal circuitry in vivo versus in vitro may have also contributed to enhanced spontaneous activity in spastic mice in vivo.

No clear mechanism could be identified that would predict enhanced post-pinch discharge in some spastic neurones. Interestingly, elevated post-pinch discharge (see Fig. 9B) appears to be a similar phenomenon to the rebound AP discharge following hyperpolarizing step-currents in vitro (Fig. 3). Despite this similarity we did not find a change in the number of spastic SDH neurones expressing rebound AP discharge, or the currents associated with this property (ICa and Ih). Alternatively, a synaptic-based mechanism involving local interneurones may elevate post-pinch discharge in this small subset of SDH neurones. A clear mechanism, however, remains to be identified. Irrespective of the how elevated spontaneous activity and enhanced post-pinch discharge occurs, these in vivo recordings identified a small population of neurones exhibiting elevated activity that would have gone undetected in an approach that relied solely on in vitro recording.

Physiological significance

In the spastic mouse, glycinergic inhibition is reduced dramatically in a number of CNS regions (Hartenstein et al. 1996; Graham et al. 2003, 2006; von Wegerer et al. 2003; Molon et al. 2006). The reduction of glycinergic inhibition in the SDH is accompanied by a compensatory increase in GABAAergic inhibition (Graham et al. 2003). In this study, we found additional changes in spastic SDH neurone properties in the form of altered potassium channel function that could also supplement inhibitory mechanisms. This raises the possibility that normal sensory processing is preserved in the SDH of spastic mice because an adequate level of inhibition is maintained.

The above results indicate that, in addition to the well-described disruption of motor function, there is a complex rearrangement of signal processing mechanisms in a sensory CNS region in spastic mice. The prominent finding from our in vitro experiments was a change in potassium channel function capable of altering intrinsic membrane and discharge properties. Particularly, there was a prominent rise in delayed firing, which we have previously shown reduces SDH neurone responses during pinch stimulation (Graham et al. 2007a). This suggests that contrary to the well-documented increase in motoneurone output after dorsal root stimulation (Biscoe & Duchen, 1986) of spastic mice, many SDH neurones decrease their output. We did not, however, detect significant changes to signal processing in the SDH of spastic mice in vivo, suggesting the overall function of this region is perhaps surprisingly preserved. Nociceptive input into the spinal cord mediates a number of functions including transmission of information related to tissue damage to various higher brain centres and reflexive withdrawal from harmful stimuli. Thus, one function of reducing the responsiveness of some SDH neurones may be to minimize the relay of this input to an already hyperexcitable ventral horn in spastic mice where excess stimulation would only compound motor disturbances. Alternatively, if disrupted inhibition in spastic mice increases transmission of nociceptive signals, reduced SDH neurone responsiveness could supplement inhibitory mechanisms that limit nociceptive transmission to higher brain centres. Assays that measure behavioural pain sensitivity in spastic mice will help determine the physiological impact of such altered potassium channel function.

Acknowledgments

This work was supported by the National Health and Medical Research Council of Australia (Grants 980382, 276403 and 401244), the Hunter Medical Research Institute, and the University of Newcastle, Australia. We thank Dr D. G. Stuart for editorial comments.

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