Fifty two age- and litter-matched wild-type (WT) and heterozygous transgenic mice overexpressing the hSOD1^G93A mutation were utilized. This study was conducted following the guidelines of the Queensland Government Animal Research Act 2001, associated Animal Care and Protection Regulations (2002 and 2008), as well as the Australian Code for the Care and Use of Animals for Scientific Purposes, 8th Edition (National Health and Medical Research Council, 2013). The University of Queensland Anatomical Biosciences Animal Ethics Committee approved all protocols involving animal use.

Mice from four previously characterized phenotypic stages were used—(1) postnatal (P) days P8-15 (7 WT and 5 SOD1 mice), when lower MNs show early signs of intrinsic and synaptic hyper-excitability (van Zundert et al., 2008; Fogarty et al., 2015); (2) P28-25 (5 WT and 5 SOD1 mice), pre-symptomatic stage when mice do not show discernable symptoms but do show synaptic and structural changes in neurons (Sgobio et al., 2008; van Zundert et al., 2008; Ozdinler et al., 2011; Fogarty et al., 2015; Saba et al., 2015); (3) P65-75 (5 WT and 5 SOD1 mice), representing disease onset, with the earliest period of MN loss, muscle weakness, and appearance of overt symptoms (Ozdinler et al., 2011; Jara et al., 2012; Lee et al., 2013; Steyn et al., 2013) and; (4) P110-130 (8 WT and 12 SOD1 mice, a mid-disease stage where mice show marked muscle mass and loss of muscle strength, respectively (Lee et al., 2013). In our SOD1 colony, end stage disease (loss of righting reflex) occurs between 150 and 175 days (Ngo et al., 2012).

Mice were anesthetized with sodium pentobarbitone (60–80 mg/kg intraperitoneally, Vetcare) and then a heparinized needle (Sigma-Aldrich) was used to intracardially exsanguinate the animal. Whole brain, brainstem, and lumbar spinal cord tissues were incubated for 5 days at 37°C in the dark in a modified rapid Golgi-Cox solution that contained 5% potassium dichromate, 5% potassium chromate, 5% mercuric chloride, as described previously (Rutledge et al., 1969) (all chemicals Sigma-Aldrich).

Brain slices (300 μm thickness) were cut with a vibrating Zeiss Hyrax V50 microtome (Carl Zeiss) to produce off-coronal (15° rostral rotation) forebrain sections, transverse brainstem sections, and parasagittal lumbar spinal cord sections (embedded in 10% agarose block in 0.1 M phosphate buffered saline [PBS]). Serial sections in 24-well plates were sequentially incubated in 30% sucrose in 0.1 M PBS (30 min), 50% ethanol (dehydration, 5 min), 0.1 M ammonium hydroxide solution (30 min, rinsed twice in distilled water (5 min), and then immersed in aluminum sulfate-based Fujihunt photo fixer (Fuijfilm, Singapore, 30 min) while protected from light exposure. After rinsing in distilled water twice (5 min each), sections were dehydrated in ethanol (70, 90, 95, and three rinses of 100%, 5 min each) and transferred to chloroform: xylene: alcohol (CXA) solution (1:1:1 ratio, 10 min). Sections were cleared in xylene (2 × 5 min each) and mounted using DPX (Sigma-Aldrich) on Superfrost Plus (Lomb Menzel Glaser) slides. Slides were air dried and stored in the dark. We note that aged mice brain tissue required longer xylene (extra 5 min) clearing due to a higher fat content.

Dorsal striatum neurons for tracing were selected from sections between bregma ~1.9 and 0.6 mm, within the striatum, which was located lateral to the lateral ventricle and ventral and medial to the corpus callosum. Hippocampal CA1 pyramidal neurons for tracing were selected from sections between bregma ~−1.1 and −2.6 mm. A mouse brain atlas (Franklin and Paxinos, 2008) was used to help define the boundaries of these areas.

Morphological properties (dendritic branching, length and dendritic spines) of Golgi-impregnated neurons were traced with a 63x objective (NA 1.4) of an Axioskop 2 microscope (Carl Zeiss) with an automated z-stage controlled by Neurolucida™ software (MBF Bioscience Inc.) as for previous reports (Fogarty et al., 2015, 2016c; Kanjhan et al., 2015). We classified small processes as spines only if they met criteria of <3 μm length and <0.8 μm cross-sectional diameter (Harris, 1999; Fogarty et al., 2015). These tracings yielded: (i) soma (irregular ellipsoid) volume (based on cross-sectional areas as previously; Kanjhan et al., 2015); (ii) total arbor length (the summed dendritic arbor length); (iii) apical or basal length (summed single apical tree length or summed length of all basal trees); (iv) mean basal tree or mean dendritic length (the mean length of each basal or dendritic tree from the soma); (v) apical and basal reach (farthest linear distance between apical or basal dendrite ending and soma); (vi) apical and basal ramifications (number of bifurcations before dendritic termination, a measure of neuronal complexity); and (vii) apical or basal dendritic spine density (number of dendritic spines per 100 μm of dendrite), as defined in our previous study (Fogarty et al., 2015; Kanjhan et al., 2015). In total, 1.34 m of dendrite length were traced in the 404 neurons that were used in this study.

All analyzed pyramidal neurons had a minimum of three intact basal dendritic trees and an apical dendrite that reached dendritic termination without exiting the brain section (Klenowski et al., 2015). Dendritic spines were assessed along the entire apical or basal dendritic arbor, as for previous studies (Fogarty et al., 2016c). Striatal MSNs were identified as for previous studies (Rafols and Fox, 1979), with spine densities of >10 spines/μm (Klenowski et al., 2016) required for inclusion, as aspiny neurons with similar shaped dendritic trees occur in rodent striatum (Rafols and Fox, 1979). The IV and XII motor nucleus were identified with a mouse brain atlas, relative to major landmarks including the aqueduct, medial longitudinal fasciculus and central canal (Franklin and Paxinos, 2008), with MNs selected by their large soma and multipolar dendrites. Lumbar MNs were identified as large neurons with longitudinal soma lengths >25 μm and dendrites arranged rostro-caudally, as for past studies (Bellinger and Anderson, 1987).

Photomicrographs of dendritic arbors were generated from minimum intensity z-stack projections (z-step size of 2 μm between images; stack depth between 20 and 100 images) that were joined digitally to form mosaics of complete cells. The imaged area was estimated from the overlaid tracing file. The mosaics appear “tessellated” because of different individual z-stack thicknesses. Representative images of spine density were taken with a 100x objective (Carl Zeiss) image at a single focal plane.

Prism 6 (Graphpad) was used for all statistical analysis. Data are given as mean ± SEM and represent the mean of all single neurons from an individual brain, brainstem or spinal cord region per mouse i.e., the n used for statistical purposes was an individual animal as the independent variable, as consistent with previous reports using the Golgi technique (Fogarty et al., 2016c). For the striatum, CA1, and brainstem regions, a minimum of two thick sections was used. The number of individual neuronal observations that contributed to these summary data is reported in the relevant tables, with a priori exclusion criteria, namely, individual data points from neurons that were beyond two standard deviations from the mean. Data was assessed for normality using a D'Agostino and Pearson omnibus test. Two-way ANOVAs with Bonferroni's post-test were applied to data with significant differences due to genotype were accepted as statistically significant with adjusted P (adj. P) values ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, and ^****P < 0.0001. Percentage changes are reported in relation to the WT mean for significantly different age groups. The researcher performing tracings (MJF) was blind to genotype.

The soma volume, total arbor length, mean tree length, dendritic reach or dendritic ramifications of striatum MSNs in SOD1 mice did not differ from WT MSNs at any age (Table 1, Figure 1). By contrast, SOD1 MSNs showed significantly decreased dendritic spine density from P65-75 onward (Figures 1C–F,H). While dendritic spine density of SOD1 and WT striatal MSNs was not significantly different at P8-16 or P28-35 (Table 1, Figure 1H), the dendritic spine density of SOD1 MSNs was reduced by 40% at P65-75 (adj. P = 0.0082^**, Table 1, Figure 1H), and by 33% at P120 (adj. P = 0.043^*, Table 1, Figure 1H).

Spine loss in MSNs has not been reported in any ALS rodent model. Corticostriatal inputs from motor and prefrontal cortex are critical for striatal regulation of goal-directed movements (Anderson et al., 2010; Rothwell et al., 2015). TDP-43 positive-inclusions occur in the striatum of ALS patients (Zhang et al., 2008), and spine density loss and synaptic hyper-activity in the SOD1 mouse motor cortex precedes dendritic shrinkage (Jara et al., 2012; Yasvoina et al., 2013; Fogarty et al., 2015, 2016c). Thus, increased corticostriatal output may contribute to MSN synaptic dysfunction, which may be involved in extrapyramidal symptoms of ALS.

While morphological changes in motor and prefrontal cortex of SOD1 mice occurs well before any disease phenotype (Jara et al., 2012; Yasvoina et al., 2013; Fogarty et al., 2015, 2016c), entorhinal cortex pyramidal neurons of SOD1 mice are unaltered (Fogarty et al., 2016c). The hippocampal CA1 region is a major output target of the entorhinal cortex, which also processes inputs from multiple cortical areas (van Groen et al., 2003) and is associated with cognitive deficits in clinical ALS (Strong et al., 2009). We therefore measured the dendritic arbors and dendritic spine densities of hippocampal CA1 pyramidal neurons, and found that there were no differences in any morphological measurement of hippocampal CA1 pyramidal neurons of SOD1 mice compared to WT controls at any age (Table 2). This lack of hippocampal pathology in SOD1 mice accords with the rarity of cognitive dysfunction in ALS patients with SOD1 mutations (Wicks et al., 2009).

Approximately one third of ALS patients show bulbar deficits as their first symptom, evident as impaired speech or swallowing that can lead to malnutrition, choking, and death (Fujimura-Kiyono et al., 2011). Synaptic and intrinsic hyper-excitability of XII MNs occurs in neonatal SOD1 mice (van Zundert et al., 2008), and oral motor deficits also occur in ALS rodent models (Smittkamp et al., 2008). At P8-15 in SOD1 XII MNs, the total arbor length was increased by 78%, compared to WT MNs (P = 0.015^*, Table 3, Figure 2Q). This increase persisted at a lower level of 20% at P28-35 in SOD1 XII MNs, compared to WT MNs (P = 0.021^*, Table 3, Figure 2Q). By P65-75, the total arbor length of SOD1 XII MNs was the same as WT controls (P = 0.48, Table 3, Figure 2Q). By P120, total arbor length of SOD1 XII MNs was reduced by 54%, compared to WT XII MNs (P < 0.0001^****, Table 3, Figure 2Q). This pattern of changes was repeated for the mean dendritic tree length. Compared to WT XII MNs, the mean tree length of SOD1 XII MNs SOD1 XII MNs was increased by 51% at P8-15 increased (P < 0.009^**, Table 3, Figure 2R), increased by 60% at P28-35 (P < 0.0005^***, Table 3, Figure 2R), not different at P65-75 (Table 3, Fig. 2 Q), and decreased by 46% decrease at P120 (P < 0.0001^****, Table 3, Figure 2R). The soma volume of XII MNs was unchanged between genotypes at all ages studied (Table 3). There was also no significant difference in the dendritic reach or dendritic ramifications of XII MNs of SOD1 and WT mice for any age studied (Table 3).

The dendritic spine is the postsynaptic compartment for excitatory synapses (Harris, 1999), and is present on the dendrites of XII MNs in neonatal WT mice (Kanjhan et al., 2015). Compared to WT littermates, the dendritic spine density of XII MNs from SOD1 mice was increased at all ages (Table 3, Figure 2). At P8-15 in SOD1 XII MNs, SOD1 XII MN spine density was increased by 101% (P = 0.0099^**, Table 3, Figures 2I,J,S); at P28-35, spine density was increased by 122% (P = 0.0023^**, Table 3, Figures 2K,L,S); at P65-75, spine density was increased by 85% (P = 0.042^*, Table 3, Figures 2M,N,S); and at P120 (P = 0.0051^**, Table 3, Figures 2O,P,S), spine density was increased by 93%, compared to WT controls at the same ages (Table 3, Figure 2S).

As respiratory MNs (which include XII MNs) tend to degenerate later than spinal cord MNs innervating limb muscles (Haenggeli and Kato, 2002; Ngo et al., 2012; Lee et al., 2013), we determined whether the dendritic changes in XII MNs were reiterated in hindlimb MNs of the lumbar spinal cord MNs of SOD1, compared to WT mice. By contrast to the early increase in dendritic length seen in XII MNs from neonatal or pre-symptomatic SOD1 mice, the dendritic arbors of SOD1 lumbar MNs did not increase, but were decreased from P28-35 onward, compared to WT controls (Figure 3). Compared to WT controls, the total arbor length of SOD1 lumbar MNs was unchanged at P8-15 (Table 4, Figure 3R), decreased by 43% at P28-35 (P = 0.023^*, Table 4, Figure 3R), decreased by 51% at P65-75 (P = 0.0001^***, Table 4, Figure 3R), and decreased by 64% at P120 (P < 0.0001^****, Table 4, Figure 3R). This pattern was repeated for mean dendritic tree lengths of SOD1 lumbar MNs, compared to WT controls, being unchanged at P8-15 (Table 4, Figure 3S), decreased by 37% at P28-35 (P = 0.0073^**, Table 4, Figure 3S), decreased by 44% at P65-75 (P = 0.0009^***, Table 4, Figure 3S), and decreased by 65% at P120 (P < 0.0001^****, Table 4, Figure 3S). A similar pattern was seen for changes in dendritic reach, which was unchanged for P8-15 SOD1 lumbar MNs (P > 0.9999, Table 4), decreased at P28-35 by 31% (P = 0.033^*, Table 4), decreased at P65-75 by 32% (P = 0.0032^**, Table 4), and further decreased at P120 by 46% (P < 0.0001^****, Table 4), compared to WT controls at the same ages.

The branching of SOD1 lumbar MN dendrites was also reduced at later disease stages. At P8-15 and P28-35, the dendritic ramifications of SOD1 lumbar MNs were unchanged, compared to WT controls (Table 4), while dendritic ramifications decreased by 31% at P65-75 (P = 0.018^*, Table 4), and then declined further to 42% at P120 in SOD1 lumbar MNs (P < 0.0001^****, Table 4), compared to WT controls at the same ages.

Finally, SOD1 lumbar MN soma volume was unchanged for the three earliest age groups (Table 4). However, by P120, the soma volume of SOD1 lumbar MNs, was reduced by 56%, compared to WT controls (P < 0.0001^****, Table 4).

We next determined whether reduced dendritic arbors of SOD1 lumbar MNs were associated with decreases in dendritic spine density, similar to changes in motor cortex and medial prefrontal cortex pyramidal neurons of SOD1 mice (Sgobio et al., 2008; Jara et al., 2012; Fogarty et al., 2015, 2016c) or whether dendritic spines were increased, as for XII MNs. Compared to WT controls, dendritic spine density of SOD1 lumbar MNs increased by 48% at P8-15 (P = 0.037^*, Table 4, Figures 3J,K,T), then returned to control levels at P28-35 and P65-75 (Table 4, Figures 3L–O,T), and decreased by 45% at P120 compared to WT controls by (P = 0.0021^**, Table 4, Figures 3P,Q,T). Thus, both XII MNs and lumbar MNs had increased dendritic spine densities at P8-16, compared to WT controls. However, while spine density in lumbar MNs then returned to control levels at P28-25 and P65-75, and then decreased at P120, spine density remained elevated in XII MNs at all ages studied. Elevated spine density could be thus interpreted as a neuroprotective response, as lumbar MNs are beginning to die by P30-35 in SOD1 mice (Ngo et al., 2012; Vinsant et al., 2013), while XII MN loss occurs later, at P90-100 (Haenggeli and Kato, 2002).

Some motor neuron pools are relatively resistant to death in ALS, such as those innervating the external muscles of the eye (Ferrucci et al., 2010). We therefore assessed the dendritic arbor and dendritic spine densities of trochlear (IV) MNs in the brainstem. There was no morphological changes in soma volume, total arbor length, mean tree length, dendritic reach, dendritic ramifications, or dendritic spine density of IV MNs from SOD1 mice, compared to WT IV MNS, for any age studied (Table 5). This lack of morphological change in IV MNs of SOD1 mice fits with the relative sparing of IV MNs in this model (Ferrucci et al., 2010).

Dendritic vacuoles, derived from degenerating mitochondria, are prevalent in lumbar MNs from P60 onward in SOD1 mice (Kong and Xu, 1998; Vinsant et al., 2013) and in the dendrites of human Betz cells in ALS post mortem tissue (Genç et al., 2017). Similar to other previous studies of vacuolization (Rafols and Fox, 1979; Goldstein et al., 1983), we found abundant dendritic vacuolation in XII MNs and lumbar MNs, at both P65-75 and P120, compared to WT MNs, quantified on a presence/absence basis (Figure 4). For the two earlier age groups, there were no differences in the percentage of XII MNs with vacuolization (Figure 4D); however, by P65-75, the percentage of MNs with vacuolization increased by ~6-fold, compared to WT controls (P = 0.028^*, Figure 4D), and this increase persisted at P120 (P < 0.0001^**** Figure 4D). Similarly, at P8-15 and P28-35, lumbar MNs in SOD1 mice did not show an increase in vacuolization, compared to WT controls (Figure 4E), but, at P65-75, there was a ~6-fold increase in the percentage of MNs with vacuolization (P = 0.034^*, Figure 4E) and by P120, this became a 10-fold increase in the percentage of lumbar MNs with vacuolization, (P < 0.0001^**** Figure 4E). By contrast, IV MNs from SOD1 mice did not exhibit significant differences in vacuolization at any age studied, compared to WT controls (P = 0.37, Figure 4C).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.