Neuronal hyperexcitability in dystrophin-deficient mdx hippocampal neurons: the importance of interleukin-6 and GABAergic regulation

Abstract

Duchenne Muscular Dystrophy (DMD) is a severe neuromuscular disorder arising from loss of the structural protein, dystrophin. It also often presents with cognitive deficits and susceptibility to epilepsy. Expressed in neurons of the hippocampus, dystrophin plays an important role in synapse formation, specifically the post-synaptic organisation of γ-aminobutyric acid A receptors (GABA_ARs). This study explored possible interactions between interleukin (IL)-6, which is elevated in DMD, and GABA_AR signalling in cultured hippocampal neurons of dystrophic mdx mice. Immunofluorescent imaging revealed altered development of network connectivity that displayed similar characteristics to dystrophin-expressing neurons cultured in elevated levels of IL-6. Mdx neurons dependably exhibited spontaneous oscillations. Calcium (Ca²⁺) signalling was further modulated by exposure to agonists and antagonists of GABA_A and GABA_BRs. IL-6-evoked Ca²⁺ responses were enhanced by muscimol, a GABA_AR agonist, in wildtype (WT) and mdx neurons, whilst bicuculline, a GABA_AR antagonist, only suppressed IL-6-evoked Ca²⁺activity in WT neurons. The GABA_BR agonist, baclofen, enhanced IL-6-evoked Ca²⁺ responses only in mdx neurons. Our findings support dysfunctional GABAergic signalling in hippocampal neurons that lack dystrophin, resulting in aberrant neuronal network excitability. The contribution of elevated levels of IL-6 further impact upon Ca²⁺ dyshomeostasis in dystrophic neurons and may underpin cognitive changes reported in dystrophinopathies.

Keywords: Duchenne muscular dystrophy; Dystrophin; GABA; Hippocampus; Learning; Memory.

Fig. 1

Elevated IL-6 increased dystrophin-expressing synaptic connections in cultured hippocampal neurons (A) The images show immunofluorescently-labelled hippocampal neurons cultured from C57BL/6 mice. Neurons have been labelled with an antibody (MANDRA-1) that identifies both Dp427 & Dp71 isoforms of dystrophin (green staining) and the Dp427 isoform alone (red staining) and (B) red MANDRA-1 staining co-expressed with the post-synaptic marker, PSD-95 (green staining). Arrows indicated co-expression of dystrophin with the post-synaptic marker (yellow labelling). (C) The representative immunofluorescent images and data plots illustrate the intensity (corrected total cell fluorescence, CTCF) of IL-6 staining in the neuronal cell bodies and processes under control culture conditions (black circles) and in culture medium supplemented with IL-6 (1 nM, > 48 h, grey open circles). Arrows indicate punctate expression of IL-6 in the cell bodies and processes. (D) The images and data plot illustrates the number of discrete clusters of dystrophin/10 µm in the processes of neurons cultured under control conditions or in culture medium supplemented with IL-6. Arrows highlight the increased expression of dystrophin clusters in treated neurons. (E) The images and data plots show clustering of IL-6 receptor (IL-6R) labelling (green staining) at synapses (indicated by arrows) labelled with the pre-synaptic marker, synaptophysin (red staining). The merged images illustrate regions where labelling is co-expressed (yellow staining). The number of IL-6R clusters and synaptophysin-labelled synapses under control and IL-6-exposed conditions are illustrated in the pooled data. * and ***p < 0.05 and p < 0.001, respectively. Scalebars: 35 µm.

Fig. 2

Loss of dystrophin is associated with neuronal hyperexcitability (A) The brightfield and immuno-labelled images show NMDA receptor (NMDA R) expression in wildtype (WT) and mdx cultured hippocampal neurons. Scalebar: 35 µm. (B) The dot plots show pooled data of the (i) frequency, (ii) amplitude and (iii) duration (full width, half max: FWHM) of spontaneous Ca²⁺ peaks in WT (black circles) and mdx (red triangles) cultured neurons under basal conditions. (C) The representative Ca²⁺ traces illustrate increased spontaneous Ca²⁺ activity in mdx hippocampal neuronal cultures (red traces) in comparison to WT neurons (black/grey traces) and traces and (D) The data plot illustrates suppression of spontaneous neuronal activity in dystrophic mdx hippocampal neurons following incubation with the neurotoxin, tetrodotoxin (TTX). (E) The data plot illustrates enhanced Ca²⁺ responses induced by high (50 mM) K⁺ in mdx neurons. ** and ***p < 0.01 and p < 0.001, respectively.

Fig. 3

Dystrophic mdx neurons have increased numbers of interleukin (IL)-6 receptor-expressing synaptic contacts but similar Ca²⁺ responses (A) The immunofluorescent images and data plots illustrate the intensity of IL-6 staining (corrected total cell fluorescence, CTCF) in the cell bodies and processes of hippocampal neurons from wildtype (WT, black circles) and mdx (red triangles) neurons. Scalebar: 35 µm. (B) The immunofluorescent images and data plot show the number of IL-6 receptor (green staining) clusters located at synapses identified using anti-synaptophysin (red staining) /10 µm of neuronal processes in WT and mdx neurons. Yellow staining in the merged images indicates co-localisation. Scalebar: 35 µm. (C) (i) The representative traces from WT (black trace) and mdx (red trace) and the dot plots of pooled data illustrating (ii) the changes in amplitude and (iii) frequency of neuronal intracellular Ca²⁺ oscillations following application of IL-6. (D) (i) The representative traces and (ii) dot plot from WT and mdx neurons show suppression of the IL-6-evoked response in mdx neurons. *, ** and ***p < 0.05, p < 0.01 and p < 0.001, respectively.

Fig. 4

IL-6-evoked Ca²⁺ responses are enhanced in cultured WT and mdx hippocampal neurons when exposed to the GABA_A receptor agonist, muscimol (A)The representative brightfield and immuno-labelled images of cultured hippocampal neurons from wildtype (WT) and dystrophin-deficient, mdx mice illustrate expression of GABA_A receptors in the cell bodies and processes of these neurons. Scalebar: 35 µm. (B) (i) The representative Ca²⁺ traces illustrate spontaneous activity in WT (black trace) and mdx (red trace) cultured neurons in the absence, presence, and following washout of the GABA_A receptor agonist, muscimol (1 µM, 20 min). The data plots of pooled data show the relative change (from baseline) of the (ii) frequency, (iii) amplitude and (iv) duration (time between 1/2 amplitude on ascent and descent of the transient; full width, half max (FWHM)) of Ca²⁺ oscillations in mdx hippocampal neurons (red triangles) as compared to WT (black circles) neurons. (C) (i) The representative traces from WT and mdx neurons and (ii) dot plot illustrate the enhanced response to IL-6 (1 nM, 3 min) in the presence of muscimol. *, ** and ***p < 0.05, p < 0.01 and p < 0.001, respectively.

Fig. 5

IL-6-evoked Ca²⁺ responses in the presence of the GABA_A receptor antagonist, bicuculline, is suppressed in WT hippocampal neurons (A) (i) The representative Ca²⁺ traces from WT (black trace) and mdx (red trace) hippocampal neurons and the data plots show the effects of the GABA_A receptor antagonist, bicuculline (100 µM, 20 min exposure) on the relative change in (ii) frequency, (iii) amplitude and (iv) duration (time between 1/2 amplitude on ascent and descent of the transient; full width, half max (FWHM)) of Ca²⁺ oscillations. (B) (i) The representative traces and (ii) dot plot illustrate suppression of the Ca²⁺ responses to IL-6 (1 nM, 3 min) in the presence of bicuculline, only in WT neurons. *, ** and ***p < 0.05, p < 0.01 and p < 0.001, respectively.

Fig. 6

The GABA_B receptor agonist, baclofen, potentiated IL-6-evoked Ca²⁺ responses in mdx hippocampal neurons (A) The representative brightfield and immuno-labelled images of cultured hippocampal neurons from wildtype (WT) and dystrophin-deficient mdx mice illustrate expression of GABA_B receptors in the cell bodies and processes of these neurons. Scalebar: 35 µm. (B) (i) The representative Ca²⁺ traces from wildtype (WT, black traces) and mdx (red traces) cultured hippocampal neurons illustrate neuronal activity in the absence, presence, and following washout, of the GABA_B receptor agonist, baclofen (100 µM, 20 min). The data plots of pooled data show the relative change (from baseline) of the (ii) frequency, (iii) amplitude and (iv) duration (time between 1/2 amplitude on ascent and descent of the transient; full width, half max (FWHM)) of Ca²⁺ oscillations in WT (black circles) and mdx hippocampal (red triangles) neurons. (C) (i) The representative traces and (ii) dot plot illustrate the enhanced response to IL-6 (1 nM, 3 min) in the presence of baclofen in mdx neurons. (D) The dot plot illustrates suppression of the amplitude of Ca²⁺ response evoked by high K⁺ in mdx neurons after exposure to baclofen. *, ** and ***p < 0.05, p < 0.01 and p < 0.001, respectively.

Fig. 7

The GABA_B receptor antagonist, phaclofen, had no effect on IL-6-evoked Ca²⁺ responses in WT and mdx hippocampal neurons (A) (i) The Ca²⁺ traces and the data plots show the effects of the GABA_B receptor antagonist, phaclofen (100 µM, 20 min exposure), on the relative change in (ii) frequency, (iii) amplitude and (iv) duration (time between 1/2 amplitude on ascent and descent of the transient; full width, half max (FWHM)) of Ca²⁺ oscillations in WT and mdx hippocampal neurons. (B) (i) The representative traces and ii dot plot show that Ca²⁺ responses to IL-6 (1 nM, 3 min) were not altered in the presence of phaclofen in WT or mdx neurons. *p < 0.05.

Fig. 8

Mdx mice exhibit increased freezing behaviours (A) The frequency of freezing (mouse did not move for more than 2 s) in the open field was increased in mdx mice as compared to wildtype (WT) controls. Other anxiety-related behaviours such as (B) the frequency of exploration of the exposed inner zone of the open field (number of times the mouse had four paws in the central area) and (C) stress-induced faecal excretion was similar in WT and mdx comparators. Exploration and escape behaviours such as the frequency of (D) rearing (number of times the mouse reared its front paws, against the wall or mid-air) and (E) jumping (number of times the mouse jumped with all four paws in the air) was similar in both strains. (F) Compulsive behaviours, such as repetitive grooming was similar in both strains. (G) The discrimination index, calculated from a novel object recognition trial, was similar in WT and mdx mice *p < 0.05.