GABAB receptors suppress burst-firing in reticular thalamic neurons

Abstract

Burst-firing in thalamic neurons is known to play a key role in mediating thalamocortical (TC) oscillations that are associated with non-REM sleep and some types of epileptic seizure. Within the TC system the primary output of GABAergic neurons in the reticular thalamic nucleus (RTN) is thought to induce the de-inactivation of T-type calcium channels in thalamic relay (TR) neurons, promoting burst-firing drive to the cortex and the propagation of TC network activity. However, RTN neurons also project back onto other neurons within the RTN. The role of this putative negative feedback upon the RTN itself is less well understood, although is hypothesized to induce de-synchronization of RTN neuron firing leading to the suppression of TC oscillations. Here we tested two hypotheses concerning possible mechanisms underlying TC oscillation modulation. Firstly, we assessed the burst-firing behavior of RTN neurons in response to GABA_B receptor activation using acute brain slices. The selective GABA_B receptor agonist baclofen was found to induce suppression of burst-firing concurrent with effects on membrane input resistance. Secondly, RTN neurons express Ca_V3.2 and Ca_V3.3 T-type calcium channel isoforms known to contribute toward TC burst-firing and we examined the modulation of these channels by GABA_B receptor activation. Utilizing exogenously expressed T-type channels we assessed whether GABA_B receptor activation could directly alter T-type calcium channel properties. Overall, GABA_B receptor activation had only modest effects on Ca_V3.2 and Ca_V3.3 isoforms. The only effect that could be predicted to suppress burst-firing was a hyperpolarized shift in the voltage-dependence of inactivation, potentially causing lower channel availability at membrane potentials critical for burst-firing. Conversely, other effects observed such as a hyperpolarized shift in the voltage-dependence of activation of both Ca_V3.2 and Ca_V3.3 as well as increased time constant of activation of the Ca_V3.3 isoform would be expected to enhance burst-firing. Together, we hypothesize that GABA_B receptor activation mediates multiple downstream effectors that combined act to suppress burst-firing within the RTN. It appears unlikely that direct GABA_B receptor-mediated modulation of T-type calcium channels is the major mechanistic contributor to this suppression.

Keywords: T-type calcium channel; absence epilepsy; low-threshold calcium potential; thalamocortical network.

Figure 1.

Baclofen suppresses burst-firing in RTN neurons. (a) Representative current-clamp recordings from neonatal RTN neurons in acute brain slices. Left and middle panels show control and baclofen-treated neuronal responses to the varying hyperpolarizing and depolarizing current injection protocols shown in right panels. (b) Histogram represents mean data for baclofen-treated neurons (n = 8) compared with the effects of H₂O (n = 6) and baclofen perfusion with CGP35348 pretreatment (n = 9) on burst-firing threshold in control (black bars) and treated (gray bars) RTN neurons (Paired sample t-test). (c) Histogram represents the percentage change in current required to achieve threshold for burst-firing for data in (b) (ANOVA with Tukey's post-test). *p < 0.05, ***p < 0.005. Scale bars represent 20 mV and 200 ms.

Figure 2.

Baclofen induces hyperpolarization and decreases input resistance in RTN neurons. Histograms representing mean resting membrane potential (a) and input resistance (b) of RTN neurons before (black columns) and after (gray columns) baclofen (50 uM) perfusion (n = 8). (c) Representative current-clamp recordings from RTN neurons under control conditions (left panel) and in the presence of baclofen with membrane potential corrected to pre-baclofen level with constant current injection (right panel). Recordings show response of RTN neurons to same current injection protocol (lower panels). (d) Histogram represents mean data from (c) for threshold current injection to achieve bursting in control conditions and with membrane-potential corrected, baclofen-treated neurons. (e) Histogram represents the percentage change in current required to achieve burst-firing for cells where depolarizing current is used to correct membrane potential vs. cells where membrane potential is not corrected following baclofen-mediated hyperpolarization. *p < 0.05, ***p < 0.005 Paired t-test. Scale bars represent 20 mV and 200 ms.

Figure 3.

Expression and validation of GABA_B receptors. (a) Western blot of lysates from untransfected, Ca_V3.2 transfected and Ca_V3.2 plus GABA_B1b and GABA_B2 transfected HEK 293 cells, exposed to Ca_V3.2, GABA_B1b, GABA_B2 and vinculin antibodies. (b) GFP (bi), RFP (bii) and merged GFP/RFP (biii) epi-fluorescence images with a corresponding transmitted light image (biv; scale bar = 50 µM) showing expression of GABA_B1b (red) and GABA_B2 (green) reporter genes transfected into HEK 293 cells. (c) Voltage-clamp traces showing response of Kir3.2 currents expressed in HEK 293 cells in control conditions (left panels) and following baclofen (50 µM) application (right panels) in cells expressing Kir3.2 co-transfected with GABA_B1b and GABA_B2 receptors (upper panels) or Kir3.2 alone (lower panels). (d) Representative Kir3.2 current in response to a 180 ms ramp depolarization from −100 to +50 mV under control conditions (black trace) and following baclofen (50 µM) application (gray trace). (e) Histogram represents mean current density data from ramp depolarizations in cells co-expressing Kir3.2 channels, plus GABA_B1B and GABA_B2 receptors under control conditions (black columns; n = 3) and following baclofen (50µM) application (gray column; n = 3). Scale bars represent 500 pA and 50 ms. **p < 0.01.

Figure 4.

Mean data for HEK 293 cells expressing Ca_V3.2, GABA_B1b and GABA_B2 receptors. (a) Current density (n = 14), (b) current density normalized to peak current density of cell during control conditions (n = 14), (c) voltage-dependence of activation (n = 14), steady-state inactivation (n = 5), (d) tau activation (n = 14), (e) tau inactivation (n = 14). *p < 0.05.

Figure 5.

Mean data for HEK 293 cells expressing Ca_V3.3, GABA_B1b and GABA_B2 receptors. (a) Current density (n = 7), (b) current density normalized to peak current density of cell during control conditions (n = 7), (c) voltage-dependence of activation (n = 7), steady-state inactivation (n = 3), (d) tau activation (n = 7), (e) tau inactivation (n = 7). *p < 0.05.