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J Physiol (2003), 551.3, pp. 905-916
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2003.049064

Altered inhibitory synaptic transmission in superficial dorsal horn neurones in spastic and oscillator mice

B. A. Graham*, P. R. Schofield†, P. Sah‡ and R. J. Callister*

	ABSTRACT

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The spastic (spa) and oscillator (ot) mouse have naturally occurring mutations in the inhibitory glycine receptor (GlyR) and exhibit severe motor disturbances when exposed to unexpected sensory stimuli. We examined the effects of the spa and ot mutations on GlyR- and GABA_AR-mediated synaptic transmission in the superficial dorsal horn (SFDH), a spinal cord region where inhibition is important for nociceptive processing. Spontaneous mIPSCs were recorded from visually identified neurones in parasagittal spinal cord slices. Neurones received exclusively GABA_AR-mediated mIPSCs, exclusively GlyR-mediated mIPSCs or both types of mIPSCs. In control mice (wild-type and spa/+) over 40 % of neurones received both types of mIPSCs, over 30 % received solely GABA_AR-mediated mIPSCs and the remainder received solely GlyR-mediated mIPSCs. In spa/spa animals, 97 % of the neurones received exclusively GABA_Aergic or both types of mIPSCs. In ot/ot animals, over 80 % of the neurones received exclusively GABA_AR-mediated mIPSCs. GlyR-mediated mIPSC amplitude and charge were reduced in spa/spa and ot/ot animals. GABA_AR-mediated mIPSC amplitude and charge were elevated in spa/spa but unaltered in ot/ot animals. GlyR- and GABA_AR-mediated mIPSC decay times were similar for all genotypes, consistent with the mutations altering receptor numbers but not kinetics. These findings suggest the spastic and oscillator mutations, traditionally considered motor disturbances, also disrupt inhibition in a sensory region associated with nociceptive transmission. Furthermore, the spastic mutation results in a compensatory increase in GABA_Aergic transmission in SFDH neurones, a form of inhibitory synaptic plasticity absent in the oscillator mouse.

(Resubmitted 10 June 2003; accepted after revision 30 June 2003; first published online 1 July 2003)
Corresponding author R. J. Callister: School of Biomedical Sciences, Faculty of Health, The University of Newcastle, Callaghan, NSW 2308, Australia. Email: bcrjc{at}mail.newcastle.edu.au

	INTRODUCTION

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Glycine acts as an important inhibitory neurotransmitter in the spinal cord and brainstem (Legendre, 2001). Naturally occurring mutations in the glycine receptor (GlyR) have been identified in humans (Floeter & Hallett, 1993), cattle (Pierce et al. 2001) and mice (Buckwater et al. 1994; Kingsmore et al. 1994; Mülhardt, et al. 1994; Ryan et al. 1994; Saul et al. 1994). All these mutations produce a phenotype with an exaggerated 'startle' response to auditory or tactile stimuli.

Spastic (spa) and oscillator (ot) mice have naturally occurring autosomal recessive GlyR mutations. Approximately 14 days after birth, homozygotes exhibit the startle phenotype (Betz, 1992) and severe motor disturbances (Simon, 1995). The spa mutation is caused by insertion of a LINE 1 transposable element in intron 5 of the GlyR subunit gene (Kingsmore et al. 1994; Mülhardt et al. 1994). This reduces the level of normal subunit protein and decreases heteromeric 1/ receptor numbers by 70-80 % (Becker et al. 1986; White & Heller, 1982). The ot mouse has a micro-deletion in exon 8 of the GlyR 1 subunit gene and this appears to result in production of a non-functional protein (Buckwater et al. 1994).

While the biochemical, molecular and genetic basis of both mutations is well understood (Rajendra & Schofield, 1995), only one study (Biscoe & Duchen, 1986) has examined the physiological consequences of the spa mutation at intact or 'native' synaptic connections. The spa mutation results in exaggerated motoneurone discharge, presumably because inhibition in interneuronal pathways is reduced. One intriguing finding in spa has been that reduced GlyR binding in spinal cord and brainstem homogenates is accompanied by enhanced -amino-butyric acid receptor (GABA_AR) binding (White & Heller, 1982; Biscoe & Duchen, 1986), which might represent a compensatory response. However, a recent physiological study on a transgenic mouse line expressing the dominant hyperekplexia human GlyR 1 subunit 271Q (Becker et al. 2002) failed to show GABA_A compensation in presumptive motoneurones. Thus, GlyR mutations can have varying effects on GABA_AR regulation.

Glycine and GABA modulate sensory as well as motor pathways. GlyR- and GABA_AR-mediated inhibition is important for processing pain-related information in the superficial dorsal horn (SFDH: Rexed's lamina I and II; Rexed, 1952) of the spinal cord (Yaksh, 1989; Sivilotti & Woolfe, 1994). Blocking GABA_ARs and GlyRs in the SFDH leads to hypersensitivity and symptoms resembling neuropathic pain (Sivilotti & Woolfe, 1994). Recently, ligation of spinal nerves has been shown to enhance GABA_AR-mediated inhibition (Kontinen et al. 2001), suggesting that compensatory mechanisms may operate to maintain appropriate levels of inhibition in disturbed sensory pathways.

We made whole-cell voltage-clamp recordings from SFDH neurones in parasagittal spinal cord slices from wild-type, spa/+, spa/spa and ot/ot mice, and examined the effects of each mutation on glycinergic and GABA_Aergic inhibition. The proportion of SFDH neurones receiving GABA_Aergic inhibition was higher in both spa/spa and ot/ot compared to controls. Furthermore, glycinergic inhibition was decreased in both spa/spa and ot/ot, whereas GABA_Aergic inhibition was enhanced in spa/spa but not ot/ot mice. These data suggest that decreased GlyR expression can lead to compensatory increases in GABA_AR expression in the SFDH of the spinal cord.

	METHODS

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Animals

All experiments were performed on spa or ot mice (Kingsmore et al. 1994; Müldhart et al. 1994) backcrossed onto the C57/Bl6 genetic background. C57/Bl6 wild-type mice were used as controls. We used animals of both sexes aged 17-52 days. Ages for each genotype are provided in Table 1. Spastic animals were bred by mating homozygous affected females (spa/spa) with heterozygous unaffected males (spa/+). As homozygous oscillator mice die by 21 days (Buckwater et al. 1994), ot animals were bred by mating heterozygous animals. Using these breeding regimes, 50 % of the spa and 25 % of the ot progeny have the startle phenotype. Homozygous affected spa and ot animals were identified approximately 2 weeks after birth according to four criteria: constant tremor; clenching of the rear legs when picked up by the tail; an impaired righting reflex when placed on their backs; and a tendency to walk on tip-toes with an arched back when handled (Simon, 1995).

Tissue preparation

All experimental procedures were approved by the University of Newcastle Animal Care and Ethics Committee. Parasagittal spinal cord slices were prepared using methods similar to those previously described for the rat (Thomson et al. 1989; Chéry & De Koninck, 1999). Briefly, mice were anaesthetised with ketamine (100 mg kg^-1 I.P.) and decapitated. The vertebral column and surrounding tissue was isolated and immersed in ice-cold oxygenated sucrose substituted artificial cerebro-spinal fluid (S-ACSF). This solution was continually bubbled with Carbogen (95 % O₂ and 5 % CO₂) and contained (mM) 250 sucrose, 25 NaHCO₂, 10 glucose, 2.5 KCl, 1 NaH₂PO₄, 1 MgCl₂ and 2.5 CaCl₂. The lumbar enlargement of the spinal cord was removed and glued lateral side down onto a brass platform with cyanoacrylate glue (Loctite 401, Loctite, Caringbah, Australia). The platform was then placed in a chamber containing oxygenated S-ACSF and parasagittal slices (300 µm thick) were obtained using a tissue slicer (Campden Instruments, Sileby, UK). Slices were transferred to a storage chamber containing oxygenated ACSF (118 mM NaCl substituted for sucrose in S-ACSF) and allowed to recover for 1 h before electrophysiological recording.

Electrophysiology

Parasagittal slices were held in a recording chamber using nylon netting fixed to a U-shaped platinum frame (Edwards et al. 1989) and continually perfused with oxygenated ACSF. Whole-cell voltage-clamp recordings were made at room temperature (21-23 °C) from visualised SFDH neurones in the L2-L5 spinal segments, using infra-red differential interference contrast (IR-DIC) optics. Under IR-DIC optics the substantia gelatinosa (lamina II) appears as a translucent band that delineates the ventral extent of the SFDH (Fig. 1A). Patch electrodes (2-5 M resistance) were filled with an internal solution containing (mM): 130 CsCl, 10 Hepes, 10 EGTA, 1 MgCl₂, 2 ATP and 0.3 GTP (pH adjusted to 7.35 with 1 M CsOH). Spontaneous synaptic currents were recorded at a holding potential of -70 mV, amplified and filtered (2 kHz) using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA, USA). In early experiments, signals were recorded onto videotape using a VCR (Vetter, Rebersburg, PA, USA) and subsequently digitised off-line at 10 kHz (Instrutech ITC-16i, Long Island, NY, USA) using Axograph V4.6 software (Axon Instruments, Foster City, CA). In later experiments data were digitised on-line.

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Figure 1. Identification and recording from mouse SFDH neurones A, lateral border of a parasagittal slice from the lumbosacral spinal cord enlargement viewed under low-power bright field illumination. A translucent band running parallel to the right border of the slice clearly identifies the superficial dorsal horn (SFDH and arrows). All recordings were made within this translucent band. The two horizontal bands running across the image are strands of nylon netting that held the slice during recording. B, at high power, under IR-DIC optics, SFDH neurones could be clearly identified and approached with a patch pipette (top left). For each recorded neurone its maximum and minimum orthogonal diameter and distance from the edge of the SFDH (right arrow in A) was noted.

Experimental protocol

After obtaining the whole-cell configuration, series resistance and neurone input resistance were calculated based on the response to a 10 mV hyperpolarising voltage step. These values were monitored at the beginning and end of each recording session and data were rejected if values changed by more than 20 %. During whole-cell recordings series resistance was compensated at 80 %. Several classes of spontaneous miniature inhibitory postsynaptic currents (mIPSCs), which represent the postsynaptic response to spontaneous release of single vesicles of neurotransmitter (Katz, 1969), were recorded with a typical experimental proceeding as follows. First, mIPSCs were pharmacologically isolated by bath application of the AMPA-kainate antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX; 10 µM) and the sodium channel blocker tetrodotoxin (TTX; 1 µM). mIPSCs recorded under these conditions represented various combinations of GlyR- and GABA_AR-mediated mIPSCs. At least 3 min of data were acquired under these conditions before bicuculline (10 µM) was added to the bath. If the addition of bicuculline abolished all synaptic activity, the mIPSCs were classed as GABA_Aergic. If any synaptic activity remained after the addition of bicuculline, strychnine (1 µM) was subsequently applied to the bath to confirm that the remaining mIPSCs were GlyR-mediated. In all cases the addition of CNQX, TTX, bicuculline and strychnine to the bath abolished all synaptic activity (see also Fig. 2A). In some experiments, the order in which strychnine and bicuculline were added to the bath was reversed. At the completion of each experiment the size of the recorded SFDH neurone's soma and its distance from the dorsal border of the grey and white matter were noted (Fig. 1B). TTX was obtained from Alomone Laboratories (Jerusalem, Israel). All other drugs were purchased from Sigma Chemicals (St Louis, MO, USA).

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Figure 2. Identification and classification of classes of inhibition in SFDH Experiments in A show mIPSCs recorded at a membrane potential of -70 mV from three SFDH neurones each receiving a different class of inhibition. In Aa, the top trace shows mIPSCs recorded in the presence of bath-applied CNQX (10 µM) and TTX (1 µM). Adding bicuculline (10 µM) to the bath reduced mIPSC frequency (middle trace). Subsequent addition of strychnine (1 µM) abolished all remaining synaptic activity (lower trace), confirming that the mIPSCs isolated in the middle trace were GlyR-mediated. Such neurones were classified as receiving mixed inhibition. In other neurones (Ab), the addition of strychnine (1 µM) to slices already exposed to CNQX and TTX abolished all synaptic activity (lower trace). These neurones were classified as receiving exclusively glycinergic inhibition. Conversely, if the addition of bicuculline (10 µM) abolished all synaptic activity (Ac, lower trace) a neurone was classified as receiving exclusively GABAAergic inhibition. B, proportions of neurones receiving mixed, GlyR-mediated or GABAAR-mediated mIPSCs differ in the four mouse genotypes. Neurones were classified into three classes according to methods outlined above, those receiving mixed inhibition (GlyR-mediated and GABAAR-mediated), those receiving glycinergic inhibition and those receiving GABAAergic inhibition. The distribution of each neurone class is similar for wild-type and spa/+ animals (2 analysis, P = 0.56). In both mutants, the proportion of neurones receiving GABAAergic inhibition is increased while the proportion of neurones receiving exclusively glycinergic inhibition is decreased (2 analysis P < 0.05). C, plots showing the location of neurones under the three classes of inhibition described in A and B. Each neurone is represented as a single dot and its location is plotted against a scale (x-axis) that indicates the neurone's location (assessed as distance from the dorsal edge of the SFDH).

Data analysis

All classes of mIPSCs were detected and captured using a sliding template method (semi-automated procedure within Axograph software package (Clements & Bekkers, 1997)). Captured mIPSCs were individually inspected and accepted for analysis according to the following criteria: (1) records that included overlapping mIPSCs were rejected; and (2) records that did not have a stable baseline before the rise or after the decay phase of the mIPSC were rejected. Data were also rejected if any significant time-dependent trend was evident in either mIPSC amplitude or instantaneous frequency over the course of the experiment (Callister & Walmsley, 1996). Analyses were performed on averaged mIPSCs, generated by aligning the rising phase of all accepted mIPSCs. Peak amplitude, rise time (calculated over 10-90 % of peak amplitude), and decay time constant (calculated over 20-80 % of the decay phase) were obtained using automated procedures within Axograph. Both GlyR- and GABA_AR-mediated mIPSCs were best fitted by a single decay time constant (see Fig. 3C). The mean synaptic charge for mIPSCs was modelled in Axograph using averages for peak amplitude, rise time and decay time.

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Figure 3. GlyR- and GABAA R-mediated mIPSCs have different decay times in control mice A shows 12 s of pharmacologically isolated GlyR-mediated mIPSCs (top two traces: recorded in CNQX (10 µM), TTX (1 µM) and bicuculline (10 µM)) and GABAAR-mediated mIPSCs (bottom two traces; recorded in CNQX (10 µM), TTX (1 µM) and strychnine (1 µM)) from a wild-type mouse. B, examples of GlyR- and GABAAR-mediated mIPSCs (captured from experiments depicted in A) showing their variable amplitudes and differing decay times. C, averaged GlyR- and GABAAR-mediated mIPSC (average of 20 records) scaled to the same peak amplitude for mIPSCs captured in A. The averaged mIPSCs have similar rise times (approximately 1 ms), but the GlyR-mediated mIPSC has a significantly faster decay time constant (10.8 vs. 29.4 ms; see also Table 2). Both mIPSCs are best fitted by a single decay time constant.

In order to assess the potential effects of electrotonic architecture on mIPSC amplitudes (Ulrich & Lüscher, 1993), we constructed mIPSC amplitude vs. rise times plots (Fig. 4A and Fig. 5A). Very weak but similar negative correlations between mIPSC amplitude and rise time existed for both types of mIPSC in control, spa/spa and ot/ot. Such weak correlations have been reported for GABA_Aergic and GlyR-mediated mIPSCs in rat SFDH neurones (Chéry & De Koninck, 1999). This analysis indicates that the influence of electrotonic architecture is similar for all genotypes and would not contribute significantly to any observed differences in mIPSC amplitudes between mutant and control animals.

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Figure 4. GlyR-mediated drive is reduced in spa/spa and ot/ot animals A, amplitude histograms for GlyR-mediated mIPSCs recorded in the presence of CNQX (10 µM), TTX (1 µM) and bicuculline (10 µM) for control and mutant animals. Because of the low mIPSC frequency in SFDH neurones, the histograms are constructed from data pooled from three neurones with similar mean amplitudes, input resistances and access resistances. Noise histograms are included (grey shaded bars) for each histogram. For each genotype, the mIPSC amplitude distributions are skewed to the right. The insets show plots of mIPSC amplitude vs. rise time. Note the same scale is used for all plots. For each genotype there are very weak but similar correlations between mIPSC amplitude and rise time (r2 values = 0.06, 0.06, 0.02 for control, spa/spa and ot/ot, respectively), indicating that electrotonic architecture of the recorded neurones does not contribute substantially to the measured mIPSC amplitudes. B, cumulative probability plots for the data in A. Data for control mice are represented by the bold continuous line, while the thin and dashed lines represent data for spa/spa and ot/ot, respectively. The mIPSC amplitude distribution in control mice differs from those in both mutants where there is a consistent shift to the left (smaller amplitudes) at all mIPSC amplitudes. C compares mean GlyR-mediated mIPSC charge for controls and each mutant. Columns represent mean ± S.E.M. from the indicated number of neurones. For each neurone, mIPSC charge (the area under a modelled mIPSC waveform) was calculated using an mIPSC constructed with an amplitude, rise time and decay time constant representative of mean values for the neurone. Asterisks indicate significant differences from control. Mean GlyR-mediated inhibition is significantly reduced (P < 0.05) in spa/spa and ot/ot animals compared to wild-type controls.

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Figure 5. GABAAR-mediated drive is increased in spa/spa and unchanged in ot/ot animals A, amplitude histograms for GABAAR-mediated mIPSCs recorded in the presence of CNQX (10 µM), TTX (1 µM) and strychnine (1 µM) for wild-type and mutant animals. Because of the low mIPSC frequency in SFDH neurones, the histograms are constructed from data pooled from three neurones with similar mean amplitudes, input resistances and access resistances. Noise histograms are included (grey shaded bars) for each histogram. For each genotype, the mIPSC amplitude distributions are skewed to the right. The insets show plots of GABAAR-mediated mIPSC amplitude vs. rise time. Note the same scale is used for all plots. For each genotype, similar weak negative correlations exist between mIPSC amplitude and rise time (r2 values are 0.01, 0.04, 0.05 for control, spa/spa and ot/ot, respectively), indicating that electrotonic architecture of the recorded neurones does not contribute substantially to the measured mIPSC amplitudes. B, cumulative probability plots for the data in A. Data for control mice are represented by the thick line. The thin and dashed lines represent data for spa/spa and ot/ot, respectively. The distributions differ in the number of GABAAR-mediated mIPSCs with amplitudes greater than 50 pA. In spa/spa mice (thin line), 20 % of the mIPSCs exceed 50 pA, whereas in control (thick line) and ot/ot mice (dashed line) less than 5 % exceed this value. C compares mean GABAAR-mediated mIPSC charge for each genotype. Columns and error bars represent mean ± S.E.M. from the indicated number of neurones. For each neurone, mIPSC charge (the area under a modelled mIPSC waveform) was calculated using a mIPSC constructed with an amplitude, rise time and decay time constant representative of mean values for the neurone. Asterisk indicates significant difference from control and ot/ot. Mean GABAAR-mediated inhibition is similar in controls and ot/ot, but significantly increased (P < 0.05) in spa/spa.

In recordings from all four genotypes it was obvious that extremely small mIPSCs could escape detection because they could not be resolved from background recording noise. This could potentially introduce errors when counting mIPSCs, and comparing mIPSC properties across genotypes. In order to assess the effect of these detection problems we constructed histograms from 5 ms of baseline that preceded each captured mIPSC. In all cases the noise distributions were Gaussian (see Fig. 4A and Fig. 5A) with means clustered around 0 pA. Their means were not different across genotypes (multiple paired t tests P < 0.01), suggesting that the effect of noise was the same for each genotype. We cannot, however, exclude the possibility that some very small mIPSCs (< 7.5 pA) may have gone undetected and were more frequent in one particular genotype.

The following statistical analyses were performed. ² analysis was used to determine if the proportion of neurones receiving the three classes of inhibition differed across mouse genotypes. Multivariate ANOVAs with one between factor (mouse genotype) were used to determine if the properties of GlyR- and GABA_AR-mediated mIPSCs differed across mouse genotypes. Student- Newman-Keuls post hoc tests were used to determine where differences among mouse genotypes existed. Finally, univariate ANOVAs were used to assess the effect of mIPSC type (glycinergic or GABA_Aergic) and mouse genotype on mIPSC characteristics. Statistical significance was set at P < 0.05. All data are expressed as means ± S.E.M.

	RESULTS

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Sample population

Data are presented for SFDH neurones obtained in four mouse genotypes: wild-type, spa/+, spa/spa and ot/ot. The diameter, location and membrane properties of these neurones for each genotype are presented in Table 1. Approximately three neurones were recorded from each animal and data from at least 32 neurones were obtained for each genotype. Pair-wise comparisons for the neurone properties presented in Table 1 revealed no significant differences across genotypes, suggesting that any differences in mIPSC characteristics were not caused by bias in neurone sampling techniques, recording conditions, or differences in the morphological and biophysical properties of SFDH neurones.

Because mIPSC characteristics can change significantly during early postnatal development (Singer et al. 1998) and animal age differs across genotypes (Table 1) we performed regression analysis for GlyR- and GABA_A-mediated mIPSC characteristics vs. age for each genotype. No significant relationship (P > 0.05) between age and mIPSC properties (rise time, amplitude or decay time) was observed for any genotype, indicating that mIPSC characteristics in mouse SFDH neurones reach adult values and remain constant for the age ranges used in this study (P17-52). We are therefore confident that our findings are not influenced by age differences for the different genotypes.

In the presence of CNQX (10 µM) and TTX (1 µM), spontaneous mIPSCs were observed in recordings from all but three SFDH neurones. In these three neurones (from oscillator mice) we could not resolve any mIPSCs, even though their morphological and membrane properties were similar to neurones listed in Table 1. They are not included in our analysis. To determine whether the mIPSCs were mediated by GlyRs, GABA_ARs or both, we next recorded mIPSCs in the presence of bicuculline or strychnine, the selective antagonists for GABA_ARs and GlyRs, respectively. The experiment shown in Fig. 2Aa is from a neurone expressing both GlyRs and GABA_ARs. The top trace shows spontaneous inhibitory mIPSCs recorded in the presence of CNQX and TTX. The middle trace shows that the addition of bicuculline (10 µM) decreased mean mIPSC amplitude (39.8 ± 0.8 pA vs. 27.4 ± 0.9 pA) and frequency (1.3 Hz vs. 0.7 Hz). This indicates that some of the mIPSCs in the top trace were bicuculline sensitive and thus mediated by GABA_ARs. The mIPSCs in the middle trace are mediated by GlyRs because addition of strychnine (1 µM) abolished all synaptic activity (Fig. 2Aa, bottom trace). In other neurones, mIPSCs recorded in the presence of CNQX and TTX were completely abolished by the application of either strychnine (Fig. 2Ab) or bicuculline (Fig. 2Ac). Using these procedures, SFDH neurones could be classified as receiving both GABA_AR- and GlyR-mediated mIPSCs (hereafter called 'mixed inhibition'; Fig. 2Aa), exclusively glycinergic (Fig. 2Ab), or exclusively GABA_Aergic inhibition (Fig. 2Ac).

The percentage of neurones in each of the three classes differed significantly between genotypes (² analysis, P < 0.05) and is shown in Fig. 2B. For wild-type and spa/+ mice, neurones receiving each class of inhibition are reasonably represented and there is no significant difference in the proportion of neurones in each class for these two genotypes (² analysis, P = 0.55). In both these unaffected genotypes over 40 % of the neurones received mixed inhibition (49 % for wild-type and 43 % for spa/+), whereas neurones receiving exclusively GABA_A or glycinergic inhibition accounted for approximately 30 % (31 % for wild-type and spa/+) and 20 % (20 % for wild-type and 26 % for spa/+), respectively. In animals with the startle phenotype the proportion of neurones receiving each type of inhibition differed markedly from controls and each other. For both spa/spa and ot/ot animals, neurones under exclusively glycinergic inhibition were rarely observed (3 % and 8 %, respectively). Similarly, in both spa/spa and ot/ot significantly more neurones received exclusively GABA_Aergic inhibition (58 % vs. 31 % in spa/spa vs. wild-type and 82 % vs. 31 % in ot/ot vs. wild-type, P < 0.05). The spa/spa and ot/ot genotypes differed in the proportions of neurones classified as receiving mixed inhibition (39 % vs. 10 %, respectively).

Together, these data suggest that the organisation of inhibition in the SFDH of the phenotypically normal (wild-type and spa/+) mice is similar. For spa/spa and ot/ot, however, the impact of the GlyR mutations is to reduce the proportion of SFDH neurones receiving exclusively GlyR-mediated mIPSCs and increase the proportion of neurones receiving exclusively GABA_AR-mediated mIPSCs. Spa/spa and ot/ot differ in the proportion of neurones receiving mixed inhibition.

The location (distance from the dorsal edge of SFDH) for neurones receiving the three classes of inhibition is shown in Fig. 2C for each genotype. We found no evidence for a concentration or restriction of any particular neurone class to a specific region of the SFDH (see Table 1). This contrasts with SFDH neurones recorded in parasagittal slices from adult (30- to 60-day-old) rats (Chéry & De Koninck, 1999). In the rat, inhibition in lamina I (within the most dorsal 20-50 µm of the SFDH) is mediated almost exclusively (30/31 neurones in the above study) by GlyRs, whereas inhibition in lamina II neurones is either solely GlyR- or GABA_AR-mediated, but not both. Interestingly, in rat lamina I SFDH neurones, the addition of benzodiazepines can unmask a GABA_AR-mediated component in most mIPSCs (Chéry & De Koninck, 1999). The authors interpreted this finding as evidence for the existence of extrasynaptic GABA_ARs in rat lamina I neurones. It is not known if a similar organisation of synaptic vs. extrasynaptic GABA_AR populations exists in mouse SFDH.

Inhibition in SFDH neurones from unaffected (wild-type (+/+) and spa/+) mice

With the increasing use of genetically altered mice for in vitro neurophysiology the issue of controls becomes important. For instance, does the presence of one mutant allele in spa/+ mice lead to any loss of function? We therefore compared the nature of inhibition to SFDH neurones in wild-type and spa/+ mice.

The properties of GlyR- and GABA_AR-mediated mIPSCs recorded under the pharmacological conditions outlined in Fig. 2A for SFDH neurones from wild-type and spa/+ mice are compared in Table 2. When GlyR-mediated mIPSCs are pharmacologically isolated in SFDH neurones by adding bicuculline (10 µM) the rise time, decay time constant, charge, and mIPSC frequency were comparable for these genotypes (P > 0.05, Table 2). Similarly, GABA_AR-mediated mIPSCs isolated by the addition of strychnine (1 µM) in wild-type and spa/+ mice have identical rise times, decay time constants, charge, and mIPSC frequency (P > 0.05). Together, these data suggest that mIPSC properties in the SFDH of wild-type and spa/+ mice are identical, and that the presence of one mutant allele does not lead to changes in mIPSC characteristics in the unaffected spa/+ mouse. We therefore pooled data from wild-type and spa/+ mice and hereafter these data are termed controls. These data are used for subsequent comparisons with spa/spa and ot/ot data.

Properties of GlyR- and GABA_AR-mediated mIPSCs in control mice

The properties of GlyR- and GABA_AR-mediated mIPSCs in SFDH neurones from control (wild-type and spa/+) mice are compared in Table 2 and Fig. 3. GlyR- and GABA_AR-mediated mIPSCs had similar rise times (0.9 ± 0.1 ms vs. 1.2 ± 0.1 ms, P > 0.05), but differed significantly in mean amplitude (39.8 ± 3.4 pA vs. 25.0 ± 1.7 pA, P < 0.05) and decay times (10.5 ± 0.5 ms vs. 27.4 ± 1.8 ms, P < 0.05). The frequency of each type of mIPSC varied widely between neurones (0.1-1.5 Hz and 0.1-0.7 Hz for GlyR- and GABA_AR-mediated mIPSCs, respectively); however, mean rates were significantly lower for GlyR-mediated mIPSCs (0.25 ± 0.05 Hz vs. 0.42 ± 0.13 Hz, P < 0.05). Together, these data are consistent with the known differences in the channel kinetics of native Gly- and GABA_ARs in central mammalian neurones (Chéry & De Koninck, 1999; Donato & Nistri, 2000; O'Brien & Berger, 2001).

GlyR-mediated inhibition is reduced in spa/spa and ot/ot mice

Comparison of GlyR-mediated mIPSCs for the two startle mutants with controls revealed a similar reduction in amplitude for spa/spa and ot/ot mice to approximately half that in controls (20.1 ± 2.1 pA and 18.7 ± 3.3 pA vs. 39.8 ± 3.4 pA, P < 0.05; Table 3, Fig. 4A and B). The highly skewed amplitude histogram for GlyR-mediated mIPSCs in control mice (Fig. 4A) raises the possibility that large mIPSCs (> 75 pA) may have been the sole contributors to the observed differences in mean mIPSC amplitudes. Examination of the cumulative probability plot in Fig. 4B for both mutants, however, shows that mIPSC amplitude was shifted to the left for all amplitudes. Thus, the reduction in GlyR-mediated mIPSC amplitude observed in spa/spa and ot/ot probably reflects a similar proportional reduction at all glycinergic synaptic connections. GlyR-mediated mIPSC frequency was also reduced in the startle mutants to below half that of controls (0.07 ± 0.02 Hz and 0.12 ± 0.02 Hz vs. 0.25 ± 0.05 Hz, P < 0.05; Table 3). The rise- and decay times for GlyR-mediated mIPSCs from the startle mutants were similar to controls (Table 3). GlyR-mediated mIPSC charge was significantly reduced in both spa/spa and ot/ot to approximately half control values (316 ± 38 pA ms and 287 ± 27 pA ms vs. 508.9 ± 44.3 pA ms, P < 0.05; Table 3, Fig. 4C). Thus, the spa and ot mutations cause a dramatic reduction in glycinergic inhibition to SFDH neurones without significantly changing receptor kinetics.

GABA_Aergic inhibition is enhanced in spa/spa but not ot/ot mice

We next compared GABA_A-mediated mIPSCs in the two mutants to controls (summarised in Table 4 and Fig. 5). The rise- and decay times of GABA_A-mediated mIPSCs were similar in the mutants and controls. In spa/spa mice the amplitude of GABA_AR-mediated mIPSCs was significantly increased above controls (32.9 ± 3.7 pA vs. 25.0 ± 1.7 pA; P < 0.05), however, GABA_AR-mediated mIPSC amplitude in ot/ot mice was unchanged (23.0 ± 2.0 pA vs. 24.6 ± 3.2 pA; Fig. 5A and B). Unlike the differences observed for GlyR-mediated mIPSC amplitude in control and mutant animals (Fig. 4A and B), the differences in GABA_AR-mediated mIPSC amplitude in spa/spa may be due to the presence of a population of large mIPSCs (> 75 pA; Fig. 5A). Careful examination of the histograms for mIPSCs with amplitudes less that 75 pA reveal that the distributions essentially overlap. Likewise, the cumulative probability plot (Fig. 5B) shows that the spa/spa distribution differs most from that of control and ot/ot animals in the number of events with large amplitudes. Thus, the increase in GABA_AR-mediated mIPSC amplitude in spa/spa may reflect a change that only occurs in a subpopulation of synaptic connections (see Discussion). Comparison of GABA_Aergic charge in the mutant animals with controls revealed significant increases in spa/spa (1089.4 ± 131.7 pA ms vs. 776.0 ± 58.9 pA ms, P < 0.05), but not in ot/ot animals (745.2 ± 56.5 pA ms vs. 776.0 ± 58.9 pA ms, P > 0.05). Thus, enhanced GABA_Aergic inhibition occurs in spa/spa but not ot/ot mice, even though both mutants show dramatic reductions in GlyR-mediated inhibition (Fig. 4B and C).

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Our results provide physiological evidence that GlyR mutations in spastic and oscillator mice alter both the contribution as well as the magnitude of glycinergic and GABA_Aergic inhibition to neurones in a sensory region of the CNS. In spa/spa SFDH neurones, glycinergic inhibition is reduced and is accompanied by an increase in GABA_Aergic inhibition. As predicted by the nature of the ot mutation (Buckwater et al. 1994), glycinergic inhibition is reduced in ot/ot mice; however, this decrease is not accompanied by changes in GABA_Aergic inhibition. Below, we consider the mechanisms underlying altered inhibitory transmission in the mutant mice and discuss the implications of these findings for the processing of noxious inputs in the SFDH.

Inhibitory transmission in the SFDH of mice with GlyR mutations

The major objective of this study was to expand our understanding of the effects of GlyR mutations on a population of neurones involved in sensory processing. GlyR mutations alter inhibitory transmission in SFDH neurones in both spa/spa and ot/ot, but in different ways.

The spastic mutation

For spa/spa, almost all SFDH neurones receive either exclusively GABA_Aergic or mixed inhibition (Fig. 2B). This shift in the contribution of glycinergic and GABA_Aergic inhibition in SFDH neurones compared to control animals is equivocal. It is unclear whether all neurones we classified as receiving exclusively GABA_Aergic inhibition in spa/spa lacked GlyRs or that their presence could not be detected. This issue similarly impacts on the detection of GABA_ARs in neurones we classify as receiving exclusively glycinergic inhibition. As our ability to detect mIPSCs was similar for all genotypes (see noise histograms in Fig. 4 and Fig. 5), we suggest that in neurones where GlyRs are reduced there are compensatory increases in GABA_AR numbers. The compensatory increase in GABA_ARs in neurones previously classified as receiving exclusively GlyR-mediated mIPSCs may lead to some neurones in spa/spa being classed as receiving mixed inhibition. Similarly, a loss of GlyRs and the addition of GABA_ARs in neurones previously classed as receiving mixed inhibition may result in GlyR-mediated mIPSCs falling below detection and hence some neurones might be classified as receiving exclusively GABA_Aergic inhibition. Evidence for such interplay between the mechanisms that regulate GABA_ARs and GlyRs is increasing. For example, both GlyR and GABA_ARs interact with the cytosolic anchoring protein gephyrin (Feng et al. 1998; Kneussel et al. 1999) and are regulated by similar intracellular signalling mechanisms (Tapia et al. 1997). Additionally, GABA and glycine can be stored and co-released from the same synaptic vesicle (Jonas et al. 1998; O'Brien & Berger, 1999), and activate GlyRs and GABA_ARs clustered at the same postsynaptic density (Todd et al. 1996; Levi et al. 1999; Dumoulin et al. 2000).

In addition to the changed contribution of glycinergic and GABA_Aergic inhibition in spa/spa SFDH neurones (Fig. 2B) there are changes in the magnitude of inhibitory mIPSCs. The amplitude of GlyR-mediated mIPSCs is reduced, and the amplitude of GABA_AR-mediated mIPSCs is increased (Fig. 4 and Fig. 5). Immunohistochemical (White, 1985), biochemical (Becker et al. 1986) and electrophysiological studies (Callister et al. 1999) indicate that the spastic mutation reduces the number of functional GlyRs. The cumulative probability plot in Fig. 4 shows that GlyR-mediated mIPSC amplitude is shifted to the left for all mIPSC amplitudes. This suggests a proportional decrease in the number of GlyRs under all glycinergic release sites.

Previous binding studies also proposed that decreased GlyR expression in spa/spa is accompanied by a compensatory increase in the number of GABA_ARs (White & Heller, 1982). The increased amplitude of GABA_AR-mediated mIPSCs we have observed at in vitro 'native' synaptic connections in mouse SFDH neurones is also consistent with the spa mutation having a predominantly postsynaptic effect (i.e. increased GABA_A receptor numbers under release sites). The presence of a long tail of large mIPSCs on the amplitude distributions for GABA_AR-mediated events in spa/spa, however, may complicate this interpretation. Recent work at GABA_Aergic connections in the cerebellum has shown that unusually large mIPSCs (200-1500 pA), which present as a long tail on mIPSC amplitude histograms, can be generated by presynaptic calcium sparks that evoke multivesicular release (Llano et al. 2000). The large cerebellar mIPSCs feature extremely fast rise times compared to smaller mIPSCs recorded from the same neurones. The large mIPSCs (75-200 pA) we observed in spa/spa were clustered around the mean rise time (~1 ms) for GABA_AR-mediated mIPSCs (see inset Fig. 5A). Thus, on the available data, presynaptic mechanisms such as calcium spark-induced multivesicular release would not appear to be strongly implicated in GABA_A compensation. Finally, the changes we have observed in GlyR- and GABA_AR-mediated mIPSC magnitude are not accompanied by changes in mIPSC kinetics (Table 3, Fig. 4; Table 4, Fig. 5). This suggests that both GlyR and GABA_AR subunit combinations are not altered by the spastic mutation.

The oscillator mutation

The ot mutation also changes the proportions of SFDH neurones that receive the three classes of inhibition (Fig. 2B and C). Unlike spa/spa, however, we find no evidence for GABA_A compensation in ot/ot, and by extension for an interplay between the mechanisms regulating GABA_ARs and GlyRs. The reduction in GlyR numbers similarly decreases the proportion of neurones classified as receiving exclusively glycinergic or mixed inhibition (Fig. 2B) and the proportion of neurones classified under purely GABA_Aergic inhibition increases accordingly. Together these data suggest that some key signal, necessary for the upregulation of GABA_AR-mediated inhibition, is lacking in ot/ot mice.

Two features of GlyR-mediated mIPSCs in ot/ot are surprising given the current understanding of this mutation. The first is the presence of any GlyR-mediated mIPSCs in ot/ot animals. The second is that GlyR-mediated mIPSC decay times are similar to controls. As the ot/ot mutation is considered a null mutation for the GlyR 1 subunit (Kling et al. 1997), the dominant subunit in the adult form of the GlyR in rodents (Legendre, 2001), one would expect an absence of GlyRs in adult ot/ot animals. Kling et al. (1997), however, noted that some GlyR expression remained (20 % of controls) in ot/ot brainstem and spinal cord homogenates. Our data also suggest that some functional GlyRs remain in ot/ot animals and perhaps provide temporary augmentation of inhibitory transmission, after the developmentally regulated switch from fetal/juvenile (2) to adult form (1/) of the receptor (Becker et al. 1988). In rats, mIPSC decay times for fetal/juvenile GlyRs are longer than those for adult GlyRs (Singer et al. 1998). Longer GlyR-mediated mIPSC decay times would therefore be expected in ot/ot mice. This however, does not appear to be the case for our data (Table 3).

Characteristics of mIPSCs in SFDH neurones and synaptic function

SFDH neurones receive synaptic inputs from a variety of sources. These include peripheral receptors, descending projections, as well as excitatory and inhibitory inputs from local circuit neurones (Willis & Coggeshall, 1991). While our data allow us to comment on the effect of each mutation at the level of individual neurones and synaptic contacts (see above), other factors also play important roles in shaping synaptic processing in the SFDH. For example, the level of convergence of inhibitory inputs onto individual SFDH neurones may have been altered by each mutation.

In other CNS regions, changes in mIPSC amplitude and frequency are interpreted as indicators of changes in the number and composition of functional synaptic sites on a given neuronal population. For example, an increase in GABA_AR-mediated mIPSC frequency in rat supraoptic nucleus neurones during pregnancy has been interpreted as an increase in the number of functional GABA_Aergic synapses (Brussard et al. 1999). Similarly, changes in mEPSC or mIPSC frequency following persistent depression of inhibitory or excitatory inputs, in cortical (Turrigiano et al. 1999) and spinal cord neurones (Galante et al. 2000), have been interpreted as changes in the number of receptors clustered at synaptic sites and the number of functional synapses (Kilman et al. 2002). Such interpretations, however, assume that neurotransmitter release probability is unchanged by the experimentally or naturally induced changes in conditions. With these caveats in mind, the decreased GlyR-mediated mIPSC amplitude and frequency observed in both spa/spa and ot/ot suggest that each mutation reduces the size (i.e. receptor number) and number of functional glycinergic inhibitory connections. In spite of the GABA_A compensation in spa/spa, mIPSC frequency is unchanged. For ot/ot, GABA_AR-mediated mIPSC amplitude and frequency remain constant. These data suggest that the size of GABA_Aergic synaptic connections in spa/spa is increased, whereas in both mutations the number of synaptic connections is unchanged. Together these changes in inhibitory synaptic function have the potential to alter inhibitory processing of nociceptive signals in the SFDH of spa/spa and ot/ot mice.

Functional implications for inhibitory transmission in the SFDH of mice with GlyR mutations

The SFDH receives information from peripheral receptors signalling pain(Woolf & Fitzgerald, 1983) and the 'gate control theory of pain' (Melzack & Wall, 1965) emphasises inhibition's important role in processing this information, with glycine and GABA both playing important roles (Game & Lodge, 1975; Sivilotti & Woolfe, 1994; Baranauskas & Nistri, 1998; Kontinen et al. 2001).

Thus, in spastic and oscillator mice, where inhibition in the SFDH is altered, one might ask if either mutation alters pain sensitivity. The spastic mutation results in reduced glycinergic inhibition and a compensatory increase in GABA_Aergic inhibition that may maintain appropriate inhibition in the SFDH (Fig. 5). In contrast, the lethal oscillator mutation results in a net decrease in inhibition. These findings can be interpreted a number of ways. If the important factor in pain processing is adequate levels of inhibition, then spa/spa may not have an altered sensitivity to pain. Alternatively, if the temporal characteristics of inhibition (e.g. mIPSC decay time) are important, then pain sensitivity would be altered in spa/spa. In ot/ot, the net loss of inhibition suggests these animals would have an altered sensitivity to pain.

To date, no studies have investigated whether the alterations to inhibitory transmission are in fact physiologically or behaviourally relevant, and whether spa/spa and ot/ot mice have altered pain sensitivity. The recent application of in vivo patch-clamp recording in the rodent dorsal horn (Furue et al. 1999; Light & Willcockson, 1999) would assist in determining if increases in GABA_AR-mediated inhibition in spa/spa can overcome the 'reduced strength of the pain gate' that accompanies impaired GlyR-mediated transmission. Comparing similar experiments in ot/ot, where 'GABA_A compensation' is absent may also prove interesting. The behavioural consequences of GlyR mutations could be tested by applying noxious chemical or mechanical stimuli and assessing behavioural responses (Kontinen et al. 2001), or changes in synaptic signalling in in vivo spinal cord preparations (Furue et al. 1999; Light & Willcockson, 1999). Together, these investigations would provide valuable insights into the contribution of GlyRs and GABA_ARs to the processing of noxious stimuli in the SFDH.

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Acknowledgements

This work was supported by the National Health and Medical Research Council of Australia (Project grant no. 980302 to R.J.C. and P.S.; Block grant no. 993050 and Fellowship no. 157209 to P.R.S.), the Ramaciotti Foundation of Australia (no. 98030), and the University of Newcastle. Thanks also go to Robin Callister for statistical advice.

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