Additional file 1: of Inflammation and neuronal death in the motor cortex of the wobbler mouse, an ALS animal model

Comparison between Iba-1-labeled microglial cells in the motor cortex of WT mice and intense symptomatic WR mice 60 d.p.n. (A–C) Relation of activated microglial cells (red) and neurons labeled with neuronal nuclei antibody (green) in brain tissue of WT mice. (D–F) Relation of activated microglial cells (red) and neurons labeled with neuronal nuclei antibody (green) in the brain tissue of severely symptomatic WR mice. (Scale bar = 20 μm). (TIF 10223 kb

Additional file 2: of Inflammation and neuronal death in the motor cortex of the wobbler mouse, an ALS animal model

Visualization of the weak staining of Iba-1- and TNF-α-labeled microglial and caspase 3-positive neuronal cells in motor cortex tissue of WR mice 20 d.p.n. (A–C) Relation of activated microglial cells (red) and neurons labeled with neuronal nuclei antibody (green) in brain tissue of non-symptomatic WR mice. (D–F) Iba-1-labeled microglial cells (red) synthesizing the cytokine TNF-α (green). (G–H) Caspase 3-positive (red) neurons labeled with NeuN (neuronal nuclei antibody) (green). (Scale bar = 20 μm). (TIF 15107 kb

The Wobbler Mouse Model of Amyotrophic Lateral Sclerosis (ALS) Displays Hippocampal Hyperexcitability, and Reduced Number of Interneurons, but No Presynaptic Vesicle Release Impairments

<div><p>Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease. It is a fatal degenerative disease, best recognized for its debilitating neuromuscular effects. ALS however also induces cognitive impairments in as many as 50% of affected individuals. Moreover, many ALS patients demonstrate cortical hyperexcitability, which has been shown to precede the onset of clinical symptoms. The wobbler mouse is a model of ALS, and like ALS patients the wobbler mouse displays cortical hyperexcitability. Here we investigated if the neocortical aberrations of the wobbler mouse also occur in the hippocampus. Consequently, we performed extracellular field excitatory postsynaptic potential recordings in the CA1 region of the hippocampus on acute brain slices from symptomatic (P45-P60) and presymptomatic (P17-P21) wobbler mice. Significant increased excitation of hippocampal synapses was revealed by leftward shifted input/output-curves in both symptomatic and presymptomatic wobbler mice, and substantiated by population spike occurrence analyses, demonstrating that the increased synaptic excitation precedes the onset of visible phenotypic symptoms in the mouse. Synaptic facilitation tested by paired-pulse facilitation and trains in wobbler and control mice showed no differences, suggesting the absence of presynaptic defects. Immunohistochemical staining revealed that symptomatic wobbler mice have a lower number of parvalbumin positive interneurons when compared to controls and presymptomatic mice. This study reveals that the wobbler mouse model of ALS exhibits hippocampal hyperexcitability. We suggest that the hyperexcitability could be caused by increased excitatory synaptic transmission and a concomitant reduced inhibition due to a decreased number of parvalbumin positive interneurons. Thus we substantiate that wobbler brain impairments are not confined to the motor cortex, but extend to the hippocampus. Importantly, we have revealed more details of the early pathophysiology in asymptomatic animals, and studies like the present may facilitate the development of novel treatment strategies for earlier intervention in ALS patients in the future. </p> </div

Reductions in parvalbumin (PV) positive interneurons in the hippocampus of the wobbler mice.

<p>(A-D) Parvalbumin (PV) positive interneurons (illustrated by arrows) were immunohistochemically stained and counted in slices of the hippocampal formation of presymptomatic (P18-P19) wobbler mice (B) and controls (A). Symptomatic (P56) wobbler mice (D) and controls (C) were also stained. Scale bars represent 200 µm. (E) During the presymptomatic phase (P18-P19) no significant differences were found in the number of parvalbumin positive interneurons per slice in any area of the hippocampal formation between wobbler and control mice. (F) At the symptomatic phase (P56) a reduction was found in all examined areas, except in the hilus, in the wobbler mice. (G) A reduction in parvalbumin positive interneurons is seen in both wobbler mice and control mice between P18-P19 and P56; however the reduction in the wobbler mice was greater in most areas. (H) The distribution of the parvalbumin positive interneurons at the different areas are similar in wobblers and controls at the presymptomatic phase (P18-P19), and remain similar at the symptomatic phase (P56), t-test: P>0.05. (P18-19: control: n = 46 slices/4 mice, wobbler: n = 48 slices/4 mice. P56: control: n = 34 slices/4 mice, wobbler: n = 31 slices/3 mice). T-test: *P<0.05; **P<0.01. Error bars represent SEM. </p

The hippocampal circuit and experimental setup.

<p>(A) Illustration of the tri-synaptic hippocampal circuit and placement of electrodes for our electrophysiological analyses. Input from the entorhinal cortex enters the dentate gyrus via the perforant pathway forming synapses on the dentate gyrus granule cells. The granule cells project axons, the mossy fibers, to the CA3 pyramidal neurons. The axons from the CA3 cells, the Schaffer collaterals, form synapses on the dendrites of the CA1 pyramidal cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#B34" target="_blank">34</a>]. Arrows indicate the direction of the neurotransmission. In all electrophysiological experiments described in this study the stimulating and recording electrodes were placed as pictured. (B) A representative trace obtained during a recording of the fEPSP. Cursors indicate the region of the rising phase of the fEPSP used to estimate the slope of the response. The slope is linearly related to the synaptic conductance and can be used as a measure of the activation of glutamatergic receptors in the postsynaptic membrane of Schaffer collateral synapses [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#B49" target="_blank">49</a>]. An arrow indicates the partially blanked stimulation artifact resulting from the brief electrical stimulation transient applied by the bipolar stimulation electrode. The afferent fiber volley (AV) is a result of the action potentials in the population of Schaffer collaterals traveling by the recording electrode and reflects the strength of the afferent input.</p

Wobbler mice do not exhibit presynaptic impairments.

<p>(A) fEPSPs recorded during train stimulation of the Schaffer collaterals, in control mice at P17-P21. Arrows illustrate the pulses employed for the analyses: the slopes of the first three and the last three pulses (pulses 1, 2, 3, 8, 9, and 10 for the types of trains consisting of 10 pulses, and pulses 1, 2, 3, 198, 199, and 200 for the types of trains consisting of 200 pulses) from each type of train were normalized to the slope of the first pulse in the given trains. (B-E) No physiologically relevant differences were observed in short-term synaptic plasticity when comparing wobbler mice and control littermates during the presymptomatic phase (comparing B to C) or the symptomatic phase (comparing D to E). However, a few results reached statistical significance when testing the trains of the same intensity in wobbler mice against controls, as indicated by * (P<0.05) or ** (P<0.01) in the figure (C compared to B) (t-test). Note the shifts in pulse number. (P17-P21: control: n = 5 slices/4 mice, wobbler: n = 6 slices/6 mice. P45-P60: control: n = 10 slices/6 mice, wobbler: n = 9 slices/7 mice). Error bars represent SEM.</p

Synaptic depletion is normal in wobbler mice.

<p>Comparisons of the sizes of the very first and very last pulses evoked during the three consecutive trains for each stimulation protocol. Responses (pulses number 10 from the types of trains consisting of 10 pulses, and pulses number 200 from the types of trains consisting of 200 pulses) were normalized to the very first pulse in the first sweep of the three consecutive trains. No physiologically relevant differences were seen between the wobbler mice (B+D) and control littermates (A+C) during the pre-symptomatic phase (P17-P21) (A+B) or the symptomatic phase (P45-P60) (C+D). Two points of statistical significance was however reached in the results, indicated by * (P<0.05) and ** (P<0.01) in the Figure (B) (t-test). Error bars represent SEM.</p

Normal release probability in wobbler mice.

<p>(A) Representative fEPSP traces recorded during paired-pulse facilitation (PPF) performed in brain slices from a wobbler mouse (left) and a control mouse (right). (B) The fEPSP slope of the second pulse was normalized to the first pulse in the respective sweep, and in each slice, the slopes of three sweeps were averaged for each stimulation interval. No significant difference was seen in the magnitude of PPF between wobbler mice and controls (wobbler: 19 slices/8 mice and control: 18 slices/9 mice). T-test: P<0.05. Error bars represent SEM.</p

Population spikes are evoked at lower stimulation intensities, but same fEPSP size, in wobbler mice.

<p>(A) Cut-out from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#pone-0082767-g002" target="_blank">Figure 2A</a>: fEPSP traces showing the first observation of a population spike, resulting from CA1 pyramidal cell firing during I/O-curve recordings in presymptomatic (P17-P21) wobbler mice (right) and controls (left). The arrows point to what was defined as the initial population spikes (corresponding to 0.15 mA and 0.3 mA, respectively, see also arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#pone-0082767-g002" target="_blank">Fig. 2A</a>). The development of the population spikes can be appreciated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#pone-0082767-g002" target="_blank">Figure 2A</a>. (B) Cut-out from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#pone-0082767-g002" target="_blank">Figure 2B</a> showing fEPSP traces recorded during experiments in wobbler mice (right) at the symptomatic phase (P45-P60) and control mice (left), with arrows illustrating the first observed population spikes (corresponding to 0.1 mA and 0.45 mA, respectively, see also arrows in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#pone-0082767-g002" target="_blank">Fig. 2B</a>). The progression of the population spikes can be seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0082767#pone-0082767-g002" target="_blank">Figure 2B</a>. (C) The average stimulation intensity needed to evoke a population spike in P17-P21 (left) and P45-P60 (right) wobbler mice and control are illustrated by the diagrams. The small circles demonstrate the stimulation intensity distribution. (D) The fEPSP slopes corresponding to the first observation of a population spike are similar in the four groups. Small circles illustrate the individual slope measurements. (P17-P21: control: n = 10 slices/5 mice, wobbler: n = 16 slices/6 mice. P45-P60: control: n = 12 slices/7 mice, wobbler: n = 16 slices/7 mice). T-test: *P<0.05; **P<0.01. Error bars represent SEM.</p