Presynaptic GABAB receptor-mediated network excitation in the medial prefrontal cortex of Tsc2+/- mice

Abstract

The TSC1 and TSC2 tumor suppressor genes control the activity of mechanistic target of rapamycin (mTOR) pathway. Elevated activity of this pathway in Tsc2^+/- mouse model leads to reduction of postsynaptic GABA_B receptor-mediated inhibition and hyperexcitability in the medial prefrontal cortex (mPFC). In this study, we asked whether presynaptic GABA_B receptors (GABA_BRs) can compensate this shift of hyperexcitability. Experiments were performed in brain slices from adolescent wild-type (WT) and Tsc2^+/- mice. Miniature and spontaneous postsynaptic currents (m/sPSCs) were recorded from layer 2/3 pyramidal neurons in mPFC using patch-clamp technique using a Cs⁺-based intrapipette solution. Presynaptic GABA_BRs were activated by baclofen (10 µM) or blocked by CGP55845 (1 µM). Independent on genotype, GABA_BR modulators bidirectionally change miniature excitatory postsynaptic current (mEPSC) frequency by about 10%, indicating presynaptic GABA_BR-mediated effects on glutamatergic transmission are comparable in both genotypes. In contrast, frequencies of both mIPSCs and sIPCSs were suppressed by baclofen stronger in Tsc2^+/- neurons than in WT ones, whereas CGP55845 significantly increased (m/s)IPSC frequencies only in WT cells. Effects of baclofen and CGP55845 on the amplitudes of evoked (e)IPSCs confirmed these observations. These data indicate (1) that GABAergic synapses are inhibited by ambient GABA in WT but not in Tsc2^+/- slices, and (2) that baclofen shifts the E/I ratio, determined as the ratio of (m/s)EPSC frequency to (m/s)IPSC frequency, towards excitation only in Tsc2^+/- cells. This excitatory presynaptic GABA_BR-mediated action has to be taken into account for a possible medication of mental disorders using baclofen.

Keywords: Autistic spectrum disorder; E/I ratio; Hyperexcitability; MTOR; Presynaptic tonic inhibition.

Fig. 1

GABA_BR agonist-induced and antagonist-induced effects on mEPSC frequency do not depend on genotype. a Representative mEPSC traces recorded from a WT neuron demonstrate the baclofen-induced effect. b Baclofen (10 µM) significantly reduces mEPSC frequency both in WT (left) and in Tsc2^+/- (middle) neurons. Relative baclofen-induced reductions of mEPSC frequency are similar in WT and Tsc2^+/- cells (right panel). c Representative mEPSC traces recorded from Tsc2^+/- cells demonstrate the CGP55845-induced effect. d CGP55845 (1 µM) significantly increases mEPSC frequency both in WT (left) and in Tsc2^+/- (middle) cells. Relative CGP55845-induced elevations of mEPSC frequency are comparable in WT and Tsc2^+/- neurons (right panel)

Fig. 2

GABA_BR agonist-induced and antagonist-induced effects on mIPSC frequency differ in WT and Tscs2 ± cells. a Representative mIPSC traces recorded from a Tsc2^+/- neuron demonstrate the baclofen-induced effect. b Baclofen (10 µM) significantly reduces mIPSC frequency both in WT (left) and in Tsc2^+/- (middle) neurons. Relative baclofen-induced reduction of mIPSC frequency is significantly larger in Tsc2^+/- cells compared with that in WT ones (right panel). c Representative mIPSC traces recorded from a WT cell demonstrate the CGP55845-induced effect. d CGP55845 (1 µM) significantly increases mIPSC frequency in WT (left) but not in Tsc2^+/- (middle) neurons. Relative CGP55845-induced elevation of mEPSC frequency is significantly stronger in WT neurons compared with that in Tsc2^+/- cells (right panel)

Fig. 3

GABA_BR agonist-induced and antagonist-induced effects on eIPSCs. a Representative eIPSC traces recorded from a WT (top) and Tsc2^+/- (bottom) cell in control and in the presence of either baclofen (10 µM) or CGP55845 (1 µM). Traces are an average of 20 consecutive recordings. b Mean eIPSC amplitudes are not significantly different in WT and Tsc2^+/- neurons. c Baclofen reduces the mean eIPSC amplitude stronger in Tcs2 ± cells compared with WT neurons (left), whereas CGP55845 increases the mean eIPSC amplitude in WT cells but not in Tsc2^+/- neurons (right panel). d In the presence of CGP55845 (CGP), the paired-pulse ratio at 500 ms interstimulus interval (PPR₅₀₀) is reduced in WT neurons (left) but increased in Tsc2^+/- ones (right panel)

Fig. 4

GABA_BR agonist-induced and antagonist-induced effects on the E/I ratio at a single cell level. a Representative mEPSC (top) and mIPSC (bottom) traces recorded from the same Tsc2^+/- neuron in control (left) and in the presence of baclofen (10 µM, right panel). b Representative mEPSC (top) and mIPSC (bottom) traces of recorded from the same WT cell in control (left) and in the presence of CGP55845 (1 µM, CGP, right panel). c Statistical data shows that the E/I ratios are comparable in WT and Tsc2^+/- neurons in control (left). The baclofen-induced increase in the E/I ratio is more pronounced in Tsc2^+/- cells than in WT cells (middle). CGP55845 decreases the E/I ratio significantly stronger in WT neurons compared with Tsc2^+/- cells (right panel)

Fig. 5

GABA_BR agonist-induced and antagonist-induced effects on network (net-)E/I ratio. a Representative sEPSC (top) and sIPSC (bottom) traces recorded from the same Tsc2^+/- neuron in control (left) and in the presence of baclofen (10 µM, right panel). b Representative sEPSC (top) and sIPSC (bottom) traces of recorded from the same WT neuron in control (left) and in the presence of CGP55845 (1 µM, right panel). c Statistical data shows that the net-E/I ratios are comparable in WT and Tsc2^+/- neurons in control (left). The baclofen-induced increase in the net-E/I ratio is more pronounced in Tsc2^+/- cells than that in WT cells (middle). CGP55845 decreases the net-E/I ratio significantly stronger in WT neurons compared with Tsc2^+/- cells (right panel)