Different forms of glycine- and GABA(A)-receptor mediated inhibitory synaptic transmission in mouse superficial and deep dorsal horn neurons

Abstract

Background: Neurons in superficial (SDH) and deep (DDH) laminae of the spinal cord dorsal horn receive sensory information from skin, muscle, joints and viscera. In both regions, glycine- (GlyR) and GABAA-receptors (GABAARs) contribute to fast synaptic inhibition. For rat, several types of GABAAR coexist in the two regions and each receptor type provides different contributions to inhibitory tone. Recent work in mouse has discovered an additional type of GlyR, (containing alpha 3 subunits) in the SDH. The contribution of differing forms of the GlyR to sensory processing in SDH and DDH is not understood.

Methods and results: Here we compare fast inhibitory synaptic transmission in mouse (P17-37) SDH and DDH using patch-clamp electrophysiology in transverse spinal cord slices (L3-L5 segments, 23 degrees C). GlyR-mediated mIPSCs were detected in 74% (25/34) and 94% (25/27) of SDH and DDH neurons, respectively. In contrast, GABAAR-mediated mIPSCs were detected in virtually all neurons in both regions (93%, 14/15 and 100%, 18/18). Several Gly- and GABAAR properties also differed in SDH vs. DDH. GlyR-mediated mIPSC amplitude was smaller (37.1 +/- 3.9 vs. 64.7 +/- 5.0 pA; n = 25 each), decay time was slower (8.5 +/- 0.8 vs. 5.5 +/- 0.3 ms), and frequency was lower (0.15 +/- 0.03 vs. 0.72 +/- 0.13 Hz) in SDH vs. DDH neurons. In contrast, GABAAR-mediated mIPSCs had similar amplitudes (25.6 +/- 2.4, n = 14 vs. 25. +/- 2.0 pA, n = 18) and frequencies (0.21 +/- 0.08 vs. 0.18 +/- 0.04 Hz) in both regions; however, decay times were slower (23.0 +/- 3.2 vs. 18.9 +/- 1.8 ms) in SDH neurons. Mean single channel conductance underlying mIPSCs was identical for GlyRs (54.3 +/- 1.6 pS, n = 11 vs. 55.7 +/- 1.8, n = 8) and GABAARs (22.7 +/- 1.7 pS, n = 10 vs. 22.4 +/- 2.0 pS, n = 11) in both regions. We also tested whether the synthetic endocanabinoid, methandamide (methAEA), had direct effects on Gly- and GABAARs in each spinal cord region. MethAEA (5 muM) reduced GlyR-mediated mIPSC frequency in SDH and DDH, but did not affect other properties. Similar results were observed for GABAAR mediated mIPSCs, however, rise time was slowed by methAEA in SDH neurons.

Conclusion: Together these data show that Gly- and GABAARs with clearly differing physiological properties and cannabinoid-sensitivity contribute to fast synaptic inhibition in mouse SDH and DDH.

Figure 1

Location of recorded SDH and DDH neurons in lumbosacral spinal cord. The location of each recorded neuron was plotted on templates of the L3, L4, and L5 spinal cord segments. Approximately 30 neurons were recorded in each segment. For SDH neurons, recordings were obtained across the entire medio-lateral extent of the dorsal horn. For DDH neurons, recordings were concentrated in the medial two thirds of the dorsal horn because dense myelination impedes visualizing neurons in lateral DDH.

Figure 2

GlyR-mediated synaptic transmission in the SDH and DDH. A representative traces showing continuous recordings of GlyR-mediated mIPSCs (holding potential - 70 mV) in the presence of TTX (1 μm), CNQX (10 μm), and bicuculline (10 μm) from an SDH (red traces) and a DDH neuron (blue traces). Note mIPSC frequency is considerably higher in DDH neurons. B individual mIPSCs from traces in A (aligned at rise onset) showing the amplitude variability in GlyR-mediated mIPSCs recorded in both the SDH (red traces) and DDH (blue traces). Inset shows averaged mIPSCs normalised to the same amplitude (same neurons in A). Note the slower decay time of GlyR-mediated mIPSCs in SDH neurons. C overlayed histograms comparing amplitude distributions of GlyR-mediated mIPSCs in SDH (red) and DDH (blue) neurons (n = 25 neurons for SDH and DDH). In the SDH distribution, only 10% of mIPSCs have amplitudes greater than 50 pA, whereas 35% of the mIPSCs in the DDH distribution are greater than 50 pA. Inset shows data presented as cumulative probability plots. D plots comparing group data for GlyR-mediated mIPSC decay time-constant and frequency in SDH and DDH neurons. GlyR-mediated mIPSC decay time-constants were slower and mIPSC frequency was lower in SDH neurons.

Figure 3

GABA_AR-mediated synaptic transmission in the SDH and DDH. A representative traces showing continuous recordings of GABA_AR-mediated mIPSCs (holding potential - 70 mV) in the presence of TTX (1 μm), CNQX (10 μm), and strychnine (1 μm) from an SDH (red traces) and a DDH neuron (blue traces). Note similar mIPSC frequency in the neurons from the two regions. B individual mIPSCs from traces in A (aligned at rise onset) showing amplitude variability of GABA_AR-mediated mIPSCs in both SDH (red traces) and DDH (blue traces). Inset shows averaged mIPSCs normalised to the same amplitude (same neurons as in A). Note the slower decay time of GABA_AR-mediated mIPSCs in SDH neurons. C overlayed group data histograms comparing amplitude distributions for GABA_Aergic mIPSCs in SDH (red) and DDH (blue) neurons (n = 14 and 18 for SDH and DDH, respectively). The overlap of the two distributions indicates that GABA_AR-mediated mIPSC amplitudes are similar in SDH and DDH neurons. Inset shows data presented as cumulative distribution plots. D plots comparing group data for GABA_AR-mediated mIPSC decay time-constant and frequency in SDH and DDH neurons. GABA_AR-mediated mIPSC decay time-constants were significantly slower in SDH neurons, however, mIPSC frequency was similar in both regions.

Figure 4

Effect of methanandamide on GlyR-mediated synaptic transmission in SDH and DDH neurons. A plot showing GlyR-mediated mIPSC frequency (holding potential - 70 mV) in an SDH neuron during bath application of methAEA (5 μm, upper bar). mIPSC frequency declines in the presence of methAEA. Middle traces are averaged mIPSCs (n = 15) under control conditions, and after 10 minutes in methAEA. Black bars above x-axis indicate when averaged mIPSCs were obtained. Plot on right presents group data summarising proportional changes in GlyR-mediated mIPSC properties in methAEA. mIPSC frequency was reduced in methAEA. mIPSC peak amplitude, rise time, and decay time-constant were unaltered. B Effect of methAEA on GlyR-mediated mIPSCs in DDH neurons (data presented in same format as A). methAEA also reduced GlyR-mediated mIPSC frequency in DDH neurons without altering mIPSC peak amplitude, rise time, or decay time-constant. Note, each data point in left panels in A and B represent averaged instantaneous mIPSC frequency, binned in 15 s intervals.

Figure 5

Effect of methanandamide on GABA_AR-mediated synaptic transmission in SDH and DDH neurons. A Plot showing GABA_AR-mediated mIPSC frequency (holding potential - 70 mV) in an SDH neuron during bath application of methAEA (5 μm, upper bar). mIPSC frequency declines significantly in the presence of methAEA. Middle traces are averaged mIPSCs (n = 15) in control conditions and after 10 minutes in methAEA. Black bars above x-axis indicate when averaged mIPSCs were obtained. Plot on right summarizes proportional changes to GABA_AR-mediated mIPSC properties in methAEA. mIPSC frequency is reduced and rise time is slowed, however, mIPSC peak amplitude and decay time constant are not altered. B Effect of methAEA on GABA_AR-mediated mIPSCs in DDH neurons (presented in same format as A). methAEA significantly reduced GABA_AR-mediated mIPSC frequency, however, mIPSC peak amplitude, rise time, and decay time-constant were not altered in DDH neurons. Each data point on left panels in A and B represent averaged instantaneous mIPSC frequency, binned in 15 s intervals.

Figure 6

Gly- and GABA_A-subunit expression in the SDH and DDH. A plots summarising qPCR analysis for GlyR subunits. Bars represent relative expression of GlyR subunits in the SDH and DDH. The α1 and β subunits were the most highly expressed in both SDH and DDH, whereas expression of the α4 subunit was negligible. The α2 and α3 subunits were expressed at lower levels in both regions. Overall, α1, β, and α2-4 subunits were expressed at significantly different levels in both regions. Note, scale on the y-axis has been broken and expanded to facilitate comparison of subunits showing lower expression levels. B plots summarising qPCR analysis for GABA_AR subunits. Bars represent relative expression of GABA_AR subunits in the SDH and DDH. Overall, GABA_AR subunit expression was more variable than that observed for GlyR subunits. No significant differences in expression levels were identified, however, the expression profile for each subunit was similar in both SDH and DDH (ie, higher expression of α2, α3, β3, and γ2).