Plasticity of postsynaptic, but not presynaptic, GABAB receptors in SSADH deficient mice

Abstract

Succinic semialdehyde dehydrogenase (SSADH) deficiency is an autosomal-recessively inherited disorder of gamma-aminobutyrate (GABA) catabolism characterized by ataxia and epilepsy. Since SSADH is responsible for GABA break-down downstream of GABA transaminase, patients manifest high extracellular levels of GABA, as well as the GABA(B) receptor (GABA(B)R) agonist gamma-hydroxybutyrate (GHB). SSADH knockout (KO) mice display absence seizures, which progress into lethal tonic-clonic seizures at around 3weeks of age. It is hypothesized that desensitization of GABA(B)Rs plays an important role in the disease, although detailed studies of pre- and postsynaptic GABA(B)Rs are not available. We performed patch-clamp recordings from layer 2/3 pyramidal neurons in neocortical brain slices of wild-type (WT) and SSADH KO mice. Electrical stimulation of GABAergic fibers during wash in of the GABA(B)R agonist baclofen revealed no difference in presynaptic GABA(B)R mediated inhibition of GABA release between WT and SSADH KO mice. In contrast, a significant decrease in postsynaptic baclofen-induced potassium currents was seen in SSADH KO mice. This reduction was unlikely to be caused by accumulation of potassium, GABA or GHB in the brain slices, or an altered expression of regulators of G-protein signaling (RGS) proteins. Finally, adenosine-induced potassium currents were also reduced in SSADH KO mice, which could suggest heterologous desensitization of the G-protein dependent effectors, leading to a reduction in G-protein coupled inwardly rectifying potassium (GIRK) channel responses. Our findings indicate that high GABA and GHB levels desensitize postsynaptic, but not certain presynaptic, GABA(B)Rs, promoting a decrease in GIRK channel function. These changes could contribute to the development of seizures in SSADH KO mice and potentially also in affected patients.

Fig. 1. Function of presynaptic GABA_BRs in neocortical layer 2/3 pyramidal cells in WT and SSADH KO mice

(A) Whole-cell recordings of evoked IPSCs (eIPSCs) in neocortical layer 2/3 pyramidal cells using extracellular stimulation. In the WT (wild type) slice, the averaged eIPSC showed paired-pulse depression of 0.50 (amplitude of eIPSC₂ relative to eIPSC₁) at a 100 ms interval. The GABA_BR agonist baclofen (10 μM) depressed eIPSC₁ by 59%, and converted the paired-pulse depression into facilitation. (B) In the SSADH KO slice, paired-pulse depression of eIPSCs was observed. Baclofen depressed eIPSC₁ by 66%, again turning depression to facilitation. (C) Baclofen reduced the amplitude of eIPSC₁ in pyramidal cells equally in WT and SSADH KO mice. eIPSC₁ was depressed by 45.6 ± 7% in WT slices (n = 8), and by 45.9 ± 5% in SSADH KO slices (n = 6). (D) The histogram shows paired-pulse depression of eIPSC2, expressed as a ratio of eIPSC₁ (100 ms inter-pulse interval) for WT (left, n = 15) and SSADH KO (right, n = 13). Baclofen (10 μM) (filled bars) equally converted the paired-pulse ratio to facilitation in WT (left, n = 8) and SSADH KO (right, n = 6) mice, indicating similar properties of presynaptic GABA_BRs.

Fig. 2. Postsynaptic GABA_BR-mediated currents are reduced in layer 2/3 pyramidal cells in SSADH KO mice

(A-B) The GABA_BR agonist baclofen (100 μM) induced outward currents in pyramidal neurons in WT (A) and SSADH KO mice (B), which were fully blocked by GABA_B antagonist CGP55845. Note the concomitant reduction in input resistance (upper traces). The neurons were voltage-clamped at -50 mV. (C) Bar graphs representing the peak amplitude of the baclofen-induced outward currents in pyramidal cells of WT (n = 9) and SSADH KO (n = 12) mice (***: P < 0.001). (D) Concentration-response curves for baclofen are shown for pyramidal neurons of WT (squares; total n = 27) and SSADH KO (triangles; total n = 28). The mean EC₅₀ of baclofen-evoked currents were 8.11 μM and 5.09 μM in pyramidal neurons of WT and SSADH KO mice respectively.

Fig. 3. Reduced baclofen currents in SSADH KO mice are not explained by accumulation of endogenous GHB or GABA in the slice

(A-B) Representative recordings of currents induced by baclofen (100 μM) in the absence (A) or presence (B) of GHB (300 μM) in pyramidal neurons of WT mice. GHB (300 μM) did not affect the peak response to baclofen. (C) Recording of the effect of CGP55845 in pyramidal neurons of SSADH KO mice. Application of CGP55845 to a SSADH KO slice revealed little tonic activation of GABA_BRs by endogenous agonists. (D) Bar graphs represent the peak amplitude of the outward current evoked by baclofen (100 μM) in control (n = 9) and in the presence of GHB (300 μM) (n = 5) in WT slices. The rightmost bar shows the amplitude of tonic GABA_B currents (n = 5) in the absence of exogenous agonists in SSADH KO mice.

Fig. 4. Decreased responsiveness to adenosine in SSADH KO mice

(A-B) Adenosine (100 μM) induced outward currents in pyramidal neurons in WT (A) and SSADH KO mice (B). Responses to adenosine (100 μM) were smaller in SSADH KO than in WT. Note the concomitant reduction in the input resistance (upper traces). The neurons were voltage-clamped at -50 mV. (C) Bar graphs representing the average peak amplitude of adenosine-induced outward currents in pyramidal cells of WT (n = 5) and SSADH KO (n = 6) mice (*: P < 0.05). In SSADH KO, the adenosine current was 39% of WT.

Fig. 5. Reduced synaptically evoked slow GABA_B receptor IPSCs in SSADH KO mice

(A-B) Slow GABA_BR-mediated IPSCs in pyramidal neurons of WT (A) and SSADH KO (B) mice. Currents were obtained in whole-cell configuration, by increasing the extracellular stimulus intensity above threshold evoking increasing GABA_BR IPSCs. Stimuli consisted of 7 pulses at 100 Hz, and the membrane was held at -50 mV (E_K = -103 mV) to minimize a GABA_AR component (E_Cl -47 mV) (C) Bar graphs representing the amplitude of the slow IPSCs in pyramidal cells of WT (n = 12) and SSADH KO (n = 15) mice (***: P < 0.001). (D) Bar graphs representing the amplitude of the GABA_B IPSC normalized to the amplitude of the GABA_A IPSC obtained from pyramidal cells of WT (n = 11) and SSADH KO (n = 20) mice (***: P < 0.001).