Development of GABAergic and glycinergic transmission in the neonatal rat dorsal horn

Abstract

Cutaneous spinal sensory transmission appears to lack inhibitory control in the newborn spinal cord, but the properties of GABAergic and glycinergic synapses in the neonatal dorsal horn have not been characterized. Whole-cell patch-clamp recordings from rat superficial dorsal horn neurons in spinal cord slices at postnatal day 0 (P0) to P2, P6-P7, and P13-P14 revealed an age-dependent increase in the frequency of spontaneous IPSCs, which were abolished by the GABA(A) receptor (GABA(A)R) antagonist bicuculline between P0 and P7 but not at P14. GABA(A)R-mediated miniature IPSCs (mIPSCs), but not glycinergic mIPSCs, were present at birth, and GABA mIPSCs remained more frequent than glycine mIPSCs at all ages. Sciatic nerve stimulation resulted in IPSCs with both GABAergic and glycinergic components, although a larger contribution arose from GABA(A) receptors at all ages. In gramicidin perforated patch-clamp recordings, exogenous GABA applications produced depolarization in 40% of neurons at P0-P2, but the reversal potential of GABA-evoked currents (E(GABA)) was consistently more negative than action potential threshold at this age. By P6-P7, GABA evoked only membrane hyperpolarization. The GABA(B)R agonist baclofen elicited an outward current in all neurons with peak amplitudes observed by P6-P7 and abolished sciatic nerve-evoked monosynaptic glutamatergic EPSCs in all groups. The results show considerable postnatal development of inhibitory processing in the dorsal horn with GABAergic mechanisms initially dominant over glycinergic events. GABA(A)R-mediated depolarizations during the first postnatal week are likely to be important for the maturation of spinal networks but do not provide a major excitatory drive to the newborn dorsal horn.

Figure 1.

sIPSCs in the superficial dorsal horn as a function of postnatal age. A, IPSCs are evident as transient outward currents from a holding potential of 0 mV. B, sIPSC frequency increases significantly with postnatal age (*p < 0.001 compared with P0–P1; Kruskal–Wallis test). C, The mean sIPSC amplitude was not significantly different between groups.

Figure 2.

sIPSCs are predominantly mediated by GABA_ARs during the first postnatal week. A, Abolition of sIPSCs by the GABA_AR antagonist bicuculline (10 μm; Bicuc) in a P7 neuron. B, Frequency of glycinergic sIPSCs (isolated with 10 μm bicuculline) increases during the second postnatal week (^†p < 0.001; *p < 0.05; Kruskal–Wallis test). C, Application of the GlyR antagonist strychnine (0.5 μm; Strych) does not significantly affect sIPSC frequency in P0–P7 dorsal horn neurons (n = 13), consistent with a dominant role for GABA_ARs during the first week. D, Exogenous applications of glycine (1 mm) elicited outward currents in all cells examined at P0–P7 (n = 18).

Figure 3.

GlyR- and GABA_A R-mediated mIPSCs in the P0–P14 dorsal horn. A, Frequency of GABA_AR mIPSCs increases with postnatal age (*p < 0.001 compared with P0–P1; Kruskal–Wallis test). GlyR mIPSCs were not observed in any of the P0–P1 neurons tested (n = 14) but were observed by the end of the first postnatal week at a significantly lower rate than GABA_AR events (^†p < 0.001; Mann–Whitney test). B, Mean mIPSC amplitudes at different postnatal ages (*p < 0.01, **p < 0.001 compared with P0–P1 with Kruskal–Wallis test; ^†p < 0.05, ^‡p < 0.005 compared with GABA mIPSC amplitude at given age with Mann–Whitney test).

Figure 4.

Primary afferent-evoked IPSCs in the neonatal superficial dorsal horn. A, Multiphasic IPSCs were evoked from a holding potential of 0 mV via electrical stimulation of the sciatic nerve (1 mA, 1 msec, 0.033 Hz) and dissected into its composite subtypes with selective GABA_AR and GlyR antagonists. For each cell, the areas under the bicuculline- and strychnine-sensitive waveforms were calculated for a 1 sec period from the onset of the IPSC. B, Average values of the AUC for GABA_AR- and GlyR-mediated components at different postnatal ages (^†p < 0.01, ^‡p < 0.05 compared with P2 with Kruskal–Wallis test; *p < 0.05 compared with GABA_AR with Mann–Whitney test).

Figure 5.

Effect of GABA on V_m of neonatal dorsal horn neurons with intact [Cl^–]_i. A, Perforated patch-clamp recording showing example of hyperpolarization in response to GABA application (1 mm, 200 msec) in a P0 neuron at its resting membrane potential. B, GABA application results in membrane depolarization but no action potential discharge in a different P0 neuron. C–E, Plots depicting the response to GABA in individual neurons at different ages. V_rest, Resting membrane potential of the cell at the time of GABA application; V_peak in GABA, membrane voltage at the peak of the GABA response in the same neuron. There was a significant difference in the distribution of depolarizing versus hyperpolarizing responses across the different age groups (p < 0.01; χ² test).

Figure 6.

Comparison of E_GABA and action potential threshold in newborn dorsal horn neurons. A, Perforated patch-clamp recording showing currents evoked by GABA application (1 mm, 200 msec) from different holding potentials (hp). Reported holding potentials have been corrected for errors attributable to uncompensated series resistance. B, Current versus voltage plot for the same neuron illustrated in A showing an E_GABA of –64.4 mV as measured by linear regression analysis (r² = 0.95). The dotted arrow identifies the AP threshold in the same neuron. C, Average values for E_GABA and AP threshold in the sampled population of P0–P1 dorsal horn neurons (n = 11). E_GABA was more negative than AP threshold in all cells examined.

Figure 7.

Potent modulation of primary afferent-evoked inputs to the superficial dorsal horn by the GABA_BR throughout the first 2 postnatal weeks. A, Glutamatergic inputs to dorsal horn cells were elicited via electrical stimulation of the sciatic nerve and classified as monosynaptic on observing a constant latency and absence of synaptic failures with 10 Hz stimulation. Example illustrates an EPSC in a P14 neuron evoked at a threshold (300 μA, 100 μsec) and latency (14.1 msec; approximate conduction velocity of 1.7 m/sec) suggestive of an input mediated by Aδ-fibers. Bath application of baclofen (10 μm) resulted in a reversible block of the evoked EPSCs. B, The effect of baclofen on EPSC amplitude was prevented by previous treatment with the selective GABA_BR antagonist CGP 55845 (2 μm). C, Baclofen abolishes primary afferent-evoked EPSCs in all age groups (expressed as the percentage of baseline EPSC amplitude inhibited by baclofen). D,Baclofen(Bac) significantly reduces the frequency, but not the amplitude, of mEPSCs in dorsal horn neurons, consistent with a presynaptic site of action (*p < 0.005; Wilcoxon matched-pairs test).

Figure 8.

Effects of postsynaptic GABA_BR activation in dorsal horn neurons vary according to postnatal age. A, Bath application of the selective GABA_BR agonist R(+)-baclofen hydrochloride elicits a slow outward current from a holding potential of –60 mV in neonatal dorsal horn cells. B, The effect of baclofen on the holding current was completely blocked by the selective GABA_BR antagonist CGP 55845 (2 μm). C, The amplitude of the baclofen-evoked outward current was significantly greater at P6–P7 and P13–P14 than at P0 (*p < 0.01; **p < 0.001; Kruskal–Wallis test).