4-bromopropofol decreases action potential generation in spinal neurons by inducing a glycine receptor-mediated tonic conductance

Abstract

Background and purpose: Impaired function of spinal strychnine-sensitive glycine receptors gives rise to chronic pain states and movement disorders. Therefore, increased activity of glycine receptors should help to treat such disorders. Although compounds targeting glycine receptors with a high selectivity are lacking, halogenated analogues of propofol have recently been considered as potential candidates. Therefore we asked whether 4-bromopropofol attenuated the excitability of spinal neurons by promoting glycine receptor-dependent inhibition.

Experimental approach: The actions of sub-anaesthetic concentrations of propofol and 4-bromopropofol were investigated in spinal tissue cultures prepared from mice. Drug-induced alterations in action potential firing were monitored by extracellular multi-unit recordings. The effects on GABAA and glycine receptor-mediated inhibition were quantified by whole-cell voltage-clamp recordings.

Key results: Low concentrations of 4-bromopropofol (50 nM) reduced action potential activity of ventral horn neurons by about 30%, compared with sham-treated slices. This effect was completely abolished by strychnine (1 μM). In voltage-clamped neurons, 4-bromopropofol activated glycine receptors, generating a tonic current of 65 ± 10 pA, while GABAA - and glycine receptor-mediated synaptic transmission remained unaffected.

Conclusions and implications: The highest glycine levels in the CNS are found in the ventral horn of the spinal cord, a region mediating pain-induced motor reflexes and participating in the control of muscle tone. 4-Bromopropofol may serve as a starting point for the development of non-sedative, non-addictive, muscle relaxants and analgesics to be used to treat low back pain.

Figure 1

Representative recording of the effect of 4-bromopropofol (50 nM) on spontaneous action potential firing of ventral horn neurons. (A) Chemical structures of 4-bromopropofol and propofol differ with respect to the presence of a bromide atom in para-position to the hydroxyl group. (B) Action potentials, visible as the vertical deflections in the voltage traces, were monitored with an extracellular electrode. Under drug-free conditions the neuron generated action potentials at a frequency of 1.6 Hz. Application of 4-bromopropofol reduced the discharge frequency to 1.1 Hz.

Figure 2

4-bromopropofol, but not propofol, significantly decreased the discharge rate of ventral horn neurons by opening glycine receptors. In each individual experiment, action potential activity was normalized to the firing rate observed under drug-free control conditions. A normalized activity of 100% indicates that the tested drugs did not alter the discharge rate as compared with the pre-drug condition. A normalized activity of 0% stands for the total depression of action potential firing. The graph shows that 50 nM propofol (PRO; n = 30) did not decrease action potential firing as compared with sham-treated slices (n = 57). Contrastingly, 4-bromopropofol (BP) reduced neuronal activity by about 30% (n = 31). 4-bromopropofol was similarly effective in the presence and absence of the specific GABA_A receptor antagonist bicuculline (80 μM, n = 33), indicating that 4-bromopropofol did not act via GABA_A receptors. In the presence of the specific glycine antagonist strychnine (1 μM, n = 35), 4-bromopropofol failed to reduce action potential firing, indicating that the drug acted via glycine receptors. **P < 0.01, significantly different from sham; anova. Numbers of experiments are shown as insets in the columns.

Figure 3

Typical recording of inhibitory postsynaptic currents mediated via glycine receptors (A) or GABA_A receptors (B). On the right-hand side, averaged synaptic events are displayed. GABAergic synaptic events (amplitude: 62 ± 5 pA, n = 30; decay time: 25 ± 7 ms, n = 30; frequency: 14 ± 2 Hz, n = 23) had a smaller amplitude and slower decay time as compared with glycinergic synaptic currents (amplitude: 117 ± 14 pA, n = 32; decay time: 11 ± 1 ms, n = 32; frequency: 18 ± 3 Hz, n = 26).

Figure 4

Effects of propofol and 4-bromopropofol on glycine receptor- and GABA_A receptor-mediated synaptic events. (A) The frequency, decay time and amplitude of glycine receptor-mediated synaptic currents were not significantly altered by 4-bromopropofol (BP; tested by anova). However, propofol (PRO) decreased the amplitude of glycinergic currents. The latter action may explain that propofol slightly increased action potential firing of ventral horn neurons as indicated in Figure 2. (B) Neither 4-bromopropofol nor propofol altered the frequency, decay time and amplitude of GABA_A receptor-mediated events (tested by anova). Numbers of experiments are shown as insets in the columns.

Figure 5

4-bromopropofol induced a tonic current and increased current fluctuations in voltage-clamped ventral horn neurons. (A) During a whole-cell voltage-clamp recording, the membrane potential was clamped to −70 mV. Action potential-dependent synaptic events were abolished by adding TTX (1 μM) to the perfusate. In addition, GABA_A receptor-dependent conductance was blocked by bicuculline (80 μM). (B) With TTX and bicuculline still present, application of 4-bromopropofol (BP) caused a large negative shift in the baseline current and increased the noise level. (C) The corresponding all point histograms of the currents indicate that the baseline was shifted to more negative values by 4-bromopropofol. (D) The distribution of the baseline fluctuations (membrane noise) as observed before and after exposing the cells to 4-bromopropofol was well fitted with a Gaussian function. 4-bromopropofol significantly increased the SD of the fitted curves [control 8.7 ± 0.96 pA (n = 6) vs. 4-bromopropofol (n = 6) 13.3 ± 0.91 pA. ***P < 0.001, significantly different from control; t-test].

Figure 6

4-bromopropofol, but not propofol induced a large strychnine-sensitive tonic conductance. (A) A typical recording demonstrating the change in tonic current caused by strychnine upon 4-bromopropofol (BP) treatment. Note that strychnine not only shifted the baseline of the holding current, but also decreased the current noise. (B) In sham-treated slices, the strychnine-induced change in tonic current was small as compared with the change observed with 4-bromopropofol. (C) Quantification of the effects of strychnine in sham-treated slices and in slices treated either with propofol (PRO) or 4-bromopropofol. Note that the changes observed in sham-treated slices and in propofol-treated slices were almost identical, indicating that propofol did not alter the strychnine-sensitive conductance. **P < 0.01, significantly different from sham; anova. Numbers of experiments are shown as insets in the columns.

Figure 7

The expression of α1 subunits of glycine receptors did not substantially alter in samples from spinal ventral horn tissue obtained from adult, embryonic (E 14) mice, and from organotypic cultures after 10 and 20 DIC. Contrastingly, the α2 glycine receptor subunits were not detectable in adult spinal ventral horn tissue, highly expressed organotypic cultures after 10 DIC and only in small amounts in organotypic cultures after 20 DIC.