Functional Consequences of the Postnatal Switch From Neonatal to Mutant Adult Glycine Receptor α1 Subunits in the Shaky Mouse Model of Startle Disease

Abstract

Mutations in GlyR α1 or β subunit genes in humans and rodents lead to severe startle disease characterized by rigidity, massive stiffness and excessive startle responses upon unexpected tactile or acoustic stimuli. The recently characterized startle disease mouse mutant shaky carries a missense mutation (Q177K) in the β8-β9 loop within the large extracellular N-terminal domain of the GlyR α1 subunit. This results in a disrupted hydrogen bond network around K177 and faster GlyR decay times. Symptoms in mice start at postnatal day 14 and increase until premature death of homozygous shaky mice around 4-6 weeks after birth. Here we investigate the in vivo functional effects of the Q177K mutation using behavioral analysis coupled to protein biochemistry and functional assays. Western blot analysis revealed GlyR α1 subunit expression in wild-type and shaky animals around postnatal day 7, a week before symptoms in mutant mice become obvious. Before 2 weeks of age, homozygous shaky mice appeared healthy and showed no changes in body weight. However, analysis of gait and hind-limb clasping revealed that motor coordination was already impaired. Motor coordination and the activity pattern at P28 improved significantly upon diazepam treatment, a pharmacotherapy used in human startle disease. To investigate whether functional deficits in glycinergic neurotransmission are present prior to phenotypic onset, we performed whole-cell recordings from hypoglossal motoneurons (HMs) in brain stem slices from wild-type and shaky mice at different postnatal stages. Shaky homozygotes showed a decline in mIPSC amplitude and frequency at P9-P13, progressing to significant reductions in mIPSC amplitude and decay time at P18-24 compared to wild-type littermates. Extrasynaptic GlyRs recorded by bath-application of glycine also revealed reduced current amplitudes in shaky mice compared to wild-type neurons, suggesting that presynaptic GlyR function is also impaired. Thus, a distinct, but behaviorally ineffective impairment of glycinergic synapses precedes the symptoms onset in shaky mice. These findings extend our current knowledge on startle disease in the shaky mouse model in that they demonstrate how the progression of GlyR dysfunction causes, with a delay of about 1 week, the appearance of disease symptoms.

Keywords: fast decay; glycine receptor; mouse model; shaky; startle disease; β8-β9 loop.

Figure 1

Genotype of shaky mice. (A) The recognition site for the restriction enzyme HpyCH4V is disrupted by the shaky mouse mutation in exon 6; left panel. PCR genotyping of shaky (Glra1^sh/sh), wild-type (Glra1^+/+) and heterozygous (Glra1^+/sh) mice with subsequent HpyCH4V digest (right panel). (B) Sequencing chromatograms of wild-type strains C57BL6 and 129/SvJ, heterozygous Glra1^+/sh, and homozygous Glra1^sh/sh showing a c.T198C transition in exon 3 and a c.C613A transition in exon 6. Both wild-type mouse strains are shown since the shaky mutation arose in a hybrid background of C57BL6 and 129/SvJ. (C) RT-PCR analysis of GlyR α1 subunit mRNA levels in spinal cord (sc) and brain stem (bs) of wild-type (n = 4) and shaky mice (n = 4). β-actin cDNA was amplified as a reference gene to ensure equal cDNA content in all samples.

Figure 2

The neuromotor phenotype of shaky mice is due to a glycinergic defect. (A) Footprint recordings. Mice painted on their hind paws were placed on a sheet of paper in a 30 cm tunnel. Representative Glra1^+/+ and Glra1^sh/sh footprints are shown at P14 and P22. (B) Homozygous Glra1^sh/sh mice (n = 9) gain less body weight than wild-type (n = 4) and heterozygous littermates (n = 19). Survival curves of Glra1^sh/sh, Glra1^+/sh, Glra1^+/+ mice. Glra1^sh/sh mice die between weeks 3 and 6 of life. (C) Loss in body weight of heterozygous Glra1^sh/ot mice following onset of symptoms (P14), Glra1^+/+ n = 19, Glra1^+/sh n = 9, Glra1^+/ot n = 25, Glra1^sh/ot n = 10, ANOVA ^**p < 0.01, ^***p < 0.001.

Figure 3

Backcross of shaky mouse line into the spasmodic mouse line. Developmental expression of the GlyR α1 subunit in shaky mice and after backcross into the mouse line spasmodic (A–D). After backcross of shaky into the spasmodic line, the expression profile was determined in spinal cord (sc), brain stem (bs) and cortex (cx) for stages P0, P7, P14, P28, P56, P84, and P100. (A) Glra1^+/+, (B) Glra1^sh/spd, (C) heterozygous Glra1^+/spd, and (D) heterozygous Glra1^+/sh. Cortex (cx) served as negative control for GlyR α1. GlyR α1 subunit was stained with mAb2b (48 kDa), and β-Actin (46 kDa) served as a loading control (LC). Cortex samples were probed with a GlyR pan-α antibody (mAb4a) labeling other GlyR α subunits in the cortex (A,B).

Figure 4

The Q177K mutation prevents functional recovery in the zebrafish mutant Dhx37. Stacked bar diagram representing four different conditions of morpholino or mRNA injections into zebrafish larvae. MO2-dhx37 is present in all four conditions. Second lane shows additional injection of a control mRNA, third line injection of wild-type GlyR α1 subunit mRNA and fourth lane injection of GlyR α1^Q177K mRNA. Black squares demonstrate the portion of severe escape behavior of zebrafish larvae in percent (%), gray squares display the portion of the mild phenotype, and red the normal escape behavior. Animals analyzed for control condition n = 31–38, animals for GlyR α1 wild-type n = 79, GlyR α1^Q177K n = 63. P-value represents significance level with ^*p < 0.05 and ^***p < 0.001.

Figure 5

Expression level and number of motoneurons in wild-type and homozygous shaky mice. (A) Western blot analysis of GlyR α1 subunit protein expression in whole brain and spinal cord homogenates. Normalization with β-actin showed no significant differences of GlyR α1 protein levels between the two genotypes (n = 3), right image shows an example of the Western blot from two different animals of each genotype (Glra1^+/+ and Glra1^sh/sh). (B) Quantitative analysis of motoneuron numbers. The quantification was made using 3–4 animals of each genotype (Glra1^+/+, n = 4; Glra1^sh/sh, n = 3). Right images are examples of brain stem sections from Glra1^+/+ and Glra1^sh/sh with arrow heads pointing to motoneurons. Error bars represent standard deviations (S.D.).

Figure 6

The neuromotor phenotype of shaky mice improves after diazepam treatment. Overall activity of Glra1^sh/sh (n = 8) mice before and after diazepam injection is displayed as time spent on a certain activity. The following activities within a 30 min time period were counted: falling (time spent on back/side vs. upright), resting (defined as the animal sitting in one spot for more than 10 s without any activity), rearing, grooming, eating/drinking, black panel Glra1^+/+ (n = 4) and red panel Glra1^sh/sh mice. Level of significance ^**p < 0.01 and ^***p < 0.001.

Figure 7

Developmental shift of glycinergic responses in Glra1^sh/sh hypoglossal motoneurons. Whole-cell voltage-clamp recordings were obtained from hypoglossal motoneurons (HMs) held at −70 mV, with symmetrical Cl⁻ concentrations inside and outside the cell. (A) Representative traces illustrate reduced glycine current in a Glra1^sh/sh HM (red) when compared to a Glra1^+/+ HM (black), both from P18-24 mice. (B) At an earlier postnatal stage (P9-13), dose-response curves for glycine-induced currents showed no difference between HMs from Glra1^+/+ (n = 7) and Glra1^sh/sh mice (n = 8). (C) Dose-response curves of Glra1^+/+ (n = 6–11) and Glra1^sh/sh (n = 8–11) HMs from more mature mice (P18-24) with severe symptoms displayed significant divergence. The EC₅₀ of glycine was significantly increased in Glra1^sh/sh mice. (D) Typical traces from P18-24 HMs illustrate lack of tonic, strychnine (2 μM)-sensitive glycine current in mutant HMs. (E) Bar diagrams summarize loss of glycinergic tone in HMs of Glra1^sh/sh mice, as indicated by missing changes in holding current (left columns) and current variance (right columns) upon application of strychnine. P-value of significance ^*p < 0.05.

Figure 8

Stationary noise analysis of glycine-evoked whole-cell currents in wild-type and mutant HMs. (A) Example of a DC whole-cell current response (top) to glycine in a Glra1^+/+ HM (P18-24) and the corresponding AC-coupled signal (bottom). (B) Variance-mean plot where AC current noise was plotted against DC current for the traces in (A). The variance (σ²) vs. mean current (I) relationship was fitted with a parabolic function of the form: σ² = iI− I²/N to obtain the unitary current (i) and the number of channels open (N) at the peak of the current response to glycine. Power spectrum of the glycine-evoked current noise in (A) was fit with a single Lorentzian function, and the time constant was calculated from the cut-off frequency f_c according to: τ = 1/2πf_c. Points above 200 Hz have been omitted, right panel in (B). (C) Comparisons of unitary currents (P9-13 Glra1^+/+ n = 7, Glra1^sh/sh n = 5; P18-24 Glra1^+/+ n = 9, Glra1^sh/sh n = 7), channel open probability (P_open) (P9-13 Glra1^+/+ n = 4, Glra1^sh/sh n = 6; P18-24 Glra1^+/+ n = 7, Glra1^sh/sh n = 9), number of channels open at the peak current (P9-13 Glra1^+/+ n = 4, Glra1^sh/sh n = 6; P18-24 Glra1^+/+ n = 7, Glra1^sh/sh n = 9), and noise time constants (derived from power spectral density analysis) (P9-13 Glra1^+/+ n = 10, Glra1^sh/sh n = 12; P18-24 Glra1^+/+ n = 16, Glra1^sh/sh n = 15). Note that P_open was significantly lower in Glra1^sh/sh mice in the older age group compared to Glra1^+/+ control. ^*p < 0.05.

Figure 9

Developmental shift of glycinergic synaptic inhibition in Glra1^sh/sh HMs. (A) Representative traces from a Glra1^+/+ HM and a Glra1^sh/sh HM at P18-24 illustrate miniature IPSCs (mIPSCs). Recordings were performed in the presence of TTX (1 μM), KA (2 mM) and bicuculline (10 μM). The superimposed traces on the right are the averaged mIPSCs from respective neurons on the left. (B–E) Bar diagrams summarize the changes of mIPSC kinetics in Glra1^sh/sh mice. Note that a change in mIPSC amplitude was already apparent in Glra1^sh/sh mice at P9-13 when the receptor replacement has already started but neuromotor symptoms have not yet appeared. Dramatic reductions in (B) mIPSC frequency (P9-13 Glra1^+/+ n = 6, Glra1^sh/sh n = 6; P18-24 Glra1^+/+ n = 9, Glra1^sh/sh n = 9), (C) amplitudes (P9-13 Glra1^+/+ n = 6, Glra1^sh/sh n = 6; P18-24 Glra1^+/+ n = 9, Glra1^sh/sh n = 9), and (E) decay (P9-13 Glra1^+/+ n = 6, Glra1^sh/sh n = 6; P18-24 Glra1^+/+ n = 10, Glra1^sh/sh n = 9) were uniformly observed in HMs from P18-24 Glra1^sh/sh mice. (D) Rise time constants (P9-13 Glra1^+/+ n = 6, Glra1^sh/sh n = 6; P18-24 Glra1^+/+ n = 10, Glra1^sh/sh n = 9). ^*p < 0.05.