THIP selectively activates extrasynaptic α4β2Δ GABAA receptors of mouse thalamocortical neurones

Adam Brown, Murray Herd, David Balfour, Delia Belelli, Jeremy Lambert

University of Dundee, Dundee, UK

GABAA receptors (GABAARs) mediate inhibitory synaptic transmission in the mammalian brain. In certain neurones a more persistent, or ‘tonic’ form of inhibiton is present, mediated by high affinity, extrasynaptic Δ-subunit containing GABAARs. The ventrobasal nucleus (VB) of the thalamus expresses extrasynaptic α4β2Δ receptors, which are implicated in the sleep-promoting effects of the GABAAR agonist THIP (Belelli et al., 2005). Here, the contribution of these receptors to the tonic conductance of VB neurones, and to the effects of THIP was investigated.

Whole-cell patch-clamp recordings of GABAAR-mediated miniature inhibitory post synaptic currents (mIPSCs) and the tonic current were made at 35 ºC and a holding potential of -60mV from VB neurones derived from P17-P25 wild type (WT), α4-/- and Δ-/- “knockout” mice of either sex in the presence of 0.5 μM TTX and 2 mM kynurenic acid (Belelli et al., 2005). Gramicidin perforated-patch, current-clamp experiments were performed to assess the effects of local THIP application on action potential firing elicited by depolarising current injection. Data are reported as mean ± S.E.

The mIPSCs recorded from WT VB neurones had a mean rise time of 0.5 ± 0.1 ms, a peak amplitude of 79 ± 3 pA and a weighted decay time constant (τw) of 3.8 ± 0.1 ms (n=18). Deletion of the α4 or the Δ subunit had no effect on the mIPSC rise time, or peak amplitude, but produced a modest decrease of the τw (τw = 3.1 ± 0.1 ms and 3.2 ± 0.1 ms, for α4-/- and Δ-/- respectively, P<0.01). For WT VB neurones, bicuculline (30μM) induced an outward current (75 ± 16 pA, n=5), that was significantly reduced (P<0.001) for both α4-/- (7 ± 1 pA, n=4) and δ-/- (13 ± 5 pA, n=5) neurones. THIP (1 μM) had no effect on the mIPSCs across the three strains. However, for WT VB neurones, 1 μM THIP induced a large inward current (-238 ± 32 pA, n=5, P<0.01), which was antagonised by 30 μM bicuculline. The THIP-induced current was significantly reduced (P<0.001) in recordings from both α4-/- (-6 ± 2 pA, n=6) and Δ-/- (-18 ± 3 pA, n=7) neurones. Hence, in VB neurones, THIP selectively activated extrasynaptic α4β2δ cf synaptic GABAA receptors. In current-clamp recordings, the local application of THIP (1 μM) to WT VB neurones induced a transient cessation of action potential (AP) discharge (AP inter-event interval [IEI], pre-THIP = 0.20 ± 0.01s, post-THIP = 3.9 ± 0.43s, n=10, P<0.001). This effect was abolished in VB neurones derived from α4-/- (pre-THIP IEI = 0.21 ± 0.01s, post-THIP IEI = 0.21 ± 0.01s, n=8), or δ-/- mice (pre-THIP IEI = 0.20 ± 0.01s, post-THIP IEI = 0.27 ± 0.09 s, n=9). Collectively, these data further confirm that extrasynaptic α4β2Δ GABAARs mediate the inhibitory “tonic” conductance of VB neurones. Additionally, these receptors, presumably expressed peri-synaptically, may modestly contribute to the “phasic” response. Furthermore, in these VB neurones, THIP selectively activates extrasynaptic α4β2Δ GABAARs to influence neuronal excitability. Such actions are likely to contribute to the behavioural profile of THIP.

BPS A J Clark studentship to ARB; Biotechnology and Biological Sciences Research Council project grant [grant number C509923]; Dr Gregg Homanics for providing the mice

Belelli, D. et al., (2005). J. Neurosci., 25, 11513-11520