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. 2010 Jul 21;30(29):9929-38.
doi: 10.1523/JNEUROSCI.1714-10.2010.

Defective GABAergic neurotransmission and pharmacological rescue of neuronal hyperexcitability in the amygdala in a mouse model of fragile X syndrome

Jose Luis Olmos-Serrano  1 , Scott M Paluszkiewicz, Brandon S Martin, Walter E Kaufmann, Joshua G Corbin, Molly M Huntsman
Affiliations

Defective GABAergic neurotransmission and pharmacological rescue of neuronal hyperexcitability in the amygdala in a mouse model of fragile X syndrome

Jose Luis Olmos-Serrano et al. J Neurosci. .

Abstract

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by variable cognitive impairment and behavioral disturbances such as exaggerated fear, anxiety and gaze avoidance. Consistent with this, findings from human brain imaging studies suggest dysfunction of the amygdala. Underlying alterations in amygdala synaptic function in the Fmr1 knock-out (KO) mouse model of FXS, however, remain largely unexplored. Utilizing a combination of approaches, we uncover profound alterations in inhibitory neurotransmission in the amygdala of Fmr1 KO mice. We demonstrate a dramatic reduction in the frequency and amplitude of phasic IPSCs, tonic inhibitory currents, as well as in the number of inhibitory synapses in Fmr1 KO mice. Furthermore, we observe significant alterations in GABA availability, both intracellularly and at the synaptic cleft. Together, these findings identify abnormalities in basal and action potential-dependent inhibitory neurotransmission. Additionally, we reveal a significant neuronal hyperexcitability in principal neurons of the amygdala in Fmr1 KO mice, which is strikingly rescued by pharmacological augmentation of tonic inhibitory tone using the GABA agonist gaboxadol (THIP). Thus, our study reveals relevant inhibitory synaptic abnormalities in the amygdala in the Fmr1 KO brain and supports the notion that pharmacological approaches targeting the GABAergic system may be a viable therapeutic approach toward correcting amygdala-based symptoms in FXS.

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Figures

Figure 1.
Figure 1.
FMRP is expressed in the majority of interneurons in the BL. A, Schematic of a coronal view of a brain at the level of the amygdala with the BL highlighted in red. B, Image of FMRP immunostaining at P21 showing the intense and high levels of FMRP expression in the L and BL in contrast to the much lesser immunostaining in other regions of the amygdala such as the central nucleus. C–E, The majority of GAD65/67-positive neurons also show FMRP expression in the BL at P21 (arrowheads). Ce, Central nucleus of the amygdala; ec, external capsule; EnD, endopiriform nucleus; L, lateral nucleus of the amygdala; Pir, piriform cortex. Scale bars: B, E (for C–E), 10 μm.
Figure 2.
Figure 2.
Phasic and tonic inhibition are impaired in Fmr1 KO mice. A1, B1, Current-clamp traces identifying cells from WT (A1) and Fmr1 KOs (B1) as excitatory principal neurons. Repetitive AP firing in response to a 40 pA depolarizing current step is shown. A2, B2, Continuous voltage-clamp traces from the above cells showing sIPSCs recorded from these same cells in the presence of APV (50 μm) and DNQX (20 μm). C, Overlay of averaged sIPSCs from cells in A1–B2. D–G, Averaged group data, showing reductions in sIPSC and mIPSC frequency (D), amplitude (E), and inhibitory efficacy (G), as well as a significant prolongation of sIPSC weighted tau (F). Note the similarity between frequency of mIPSCs in WT and sIPSCs in Fmr1 KOs. These differences were not related to differences in intrinsic membrane properties (Vm: WT, −61.1 ± 0.7 mV; Fmr1 KO, −60.7 ± 0.8 mV; p = 0.95; Rm: WT, 223 ± 13 MΩ; Fmr1 KO, 217 ± 13 MΩ; p = 0.55). H, I, Voltage-clamp traces from representative WT and Fmr1 KO principal neurons (Vhold = −60 mV) indicating the difference between the average baseline holding current and average holding current in locally applied 100 μm Gabazine, as determined by fitted Gaussian curve (see Materials and Methods). J, Total inhibitory tonic current is significantly reduced in Fmr1 KO principal neurons. K, Voltage-clamp trace from a WT principal neuron illustrating its AP-dependent tonic GABAergic current and total tonic current. L, Averaged group data, showing that the primary source of tonic inhibitory current in both WT and Fmr1 KO principal neurons is AP-dependent GABA release. *p < 0.05; ***p < 0.005. Calibration: A1, B1, 20 mV, 200 ms; A2, B2, 100 pA, 500 ms; C, 20 pA, 20 ms; H, I, 20 pA, 5 s; K, 10 pA, 5 s.
Figure 3.
Figure 3.
GAD65/67 levels are reduced in Fmr1 KOs. A, B, Normal gross morphology and positioning of GAD65/67+ inhibitory neurons in Fmr1 KO mice (B) compared with WT (A) in the BL is shown at P21 at low-power magnification. C, D, Qualitative decrease in GAD65/67 immunostaining is observed in Fmr1 KOs compared with WT as shown at high-power magnification. E, F, Western blot analyses reveal that GAD65/67 expression levels are significantly decreased in the Fmr1 KOs, compared with WT in the BL. BL, Basolateral nucleus of the amygdala; Ce, central nucleus of the amygdala; ec, external capsule; EnD, endopiriform nucleus; L, lateral nucleus of the amygdala; Pir, piriform cortex. *p < 0.05. Scale bars: B, 100 μm; D, 10 μm.
Figure 4.
Figure 4.
Synaptic GABA release is reduced in Fmr1 KOs. A–C, mIPSC amplitude is reduced to a greater extent by TPMPA in Fmr1 KO than in WT mice. Averaged mIPSCs from individual cells from WT (A) and Fmr1 KO (B) under control conditions and with TPMPA (200 μm) (red traces) illustrate the reduction in amplitude. C, Averaged group data indicating that TPMPA had a significantly greater effect on mIPSCs in Fmr1 KOs at doses of 200 and 100 μm. This trend was also observed using a 50 μm dose, but was not statistically significant. TPMPA did not exert a significant effect on mIPSC decay. *p < 0.05. Calibration: 2 pA, 20 ms.
Figure 5.
Figure 5.
AP-dependent GABA release is impaired in Fmr1 KO mice. A–C, The PPR of evoked IPSCs is greater in excitatory principal neurons in Fmr1 KO than WT mice. Averaged traces from single cells from WT (A) and Fmr1 KO mice (B) illustrate that the strong depression observed in WT is attenuated in Fmr1 KOs. C, Averaged group data. D, Average amplitude CV of the first evoked response is also greater in Fmr1 KOs. *p < 0.05; ***p < 0.005. Calibration: 50 pA, 100 ms.
Figure 6.
Figure 6.
Reduced number of inhibitory synapses but no changes in VGAT expression in Fmr1 KOs. A–D, Quantitative analyses of electron micrographs of the BL show a remarkable decrease in inhibitory synapses in Fmr1 KOs with respect to WT. Excitatory synapses are decreased as well in Fmr1 KOs, although to a lesser extent (D). Arrowheads in A identify synaptic contacts. Detail of a symmetric (B) and asymmetric (C) synaptic contacts in the BL (arrows). E, F, Qualitative images of VGAT-immunostaining in the BL show no difference between WT (E) and Fmr1 KOs (F). G, Quantification of VGAT puncta (5 sections from 4 different mice) show no differences between WT and the Fmr1 KOs. H, I, Western blot analyses demonstrate that VGAT expression levels are similar in Fmr1 KOs and WT mice in the BL (WT, n = 4; Fmr1 KO, n = 4). *p < 0.05; ***p < 0.005. Scale bars, 20 μm.
Figure 7.
Figure 7.
THIP rescues neuronal excitability in Fmr1 KOs. A, B, Concatenated traces from single cells in response to depolarizing current steps of increasing amplitude (10 pA steps). In control ACSF (A, B1), cells from Fmr1 KO mice exhibit higher action potential firing rates for a given depolarizing current step, as well as a lower threshold for action potential generation than cells from WT mice. C, Group F–I plot illustrating hyperexcitability of cells in Fmr1 KOs. No differences in passive membrane properties were observed between cells in the two groups (Vm: WT, −61.5 ± 0.5 mV; Fmr1 KO, −61.2 ± 0.7 mV; p = 0.80; Rm: WT, 231 ± 21 MΩ; Fmr1 KO, 242 ± 12 MΩ; p = 0.19). B2, D, Bath application of THIP increases action potential threshold in Fmr1 KOs to WT levels. *p < 0.05; **p < 0.01; n.s. not statistically significant. Calibration: 40 mV, 1 s.
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