New Results

Inhibitory interneurons show early dysfunction in a SOD1 mouse model of ALS

C. F. Cavarsan, P. R. Steele, L. M. McCane, K. J. LaPre, A. C. Puritz, N. Katenka, K. A. Quinlan
doi: https://doi.org/10.1101/2020.10.21.348359

Abstract

Few studies in amyotrophic lateral sclerosis (ALS) focus on the inhibitory interneurons synapsing onto motoneurons (MNs). However, inhibitory interneurons could contribute to dysfunction, particularly if altered before MN neuropathology and establishing a long-term imbalance of inhibition / excitation. We directly assessed excitability and morphology of glycinergic (GlyT2) interneurons located throughout the ventral horn in the lumbar enlargement from SOD1G93AGlyT2eGFP (SOD1) and wildtype GlyT2eGFP (WT) mice on postnatal day 6 to 10. Patch clamp revealed dampened excitability in SOD1 interneurons, including depolarized persistent inward currents (PICs), depolarized threshold for firing action potentials, and a shortened afterhyperpolarization (AHP). SOD1 inhibitory interneurons also had smaller soma but increased dendritic volume and surface area. GlyT2 interneurons were then divided into 3 subgroups based on location: interneurons within 100 μm of the ventral white matter where Renshaw cells (RCs) are located, interneurons interspersed with MNs in lamina IX, and interneurons in the intermediate ventral area including laminae VII and VIII. Lamina IX interneurons were the most profoundly affected, including more depolarized PICs, smaller somata and larger dendritic arborization. Interneurons in lamina IX had depolarized PIC onset, smaller somata and longer dendrites. In lamina VII-VIII, interneurons were largely unaffected, mainly showing smaller somata. In summary, inhibitory interneurons show very early region-specific perturbations poised to impact excitatory / inhibitory balance of MNs, modify motor output, and provide early biomarkers of ALS. Therapeutics like riluzole which universally reduce CNS excitability could exacerbate the inhibitory dysfunction described here.

Significance Statement Spinal inhibitory interneurons could contribute to amyotrophic lateral sclerosis (ALS) pathology as they provide synaptic inhibition to spinal motoneurons (MNs). To investigate their role in the presymptomatic phase, we studied the excitability and morphology of glycinergic interneurons in transgenic mice (SOD1G93AGlyT2eGFP). We found inhibitory interneurons were less excitable and had smaller somas but larger dendrites in SOD1 mice. GlyT2 interneurons were analyzed according to their localization within the ventral spinal cord. Interestingly, the greatest differences were observed in the interneurons located in the ventral most 100 μm. We conclude that inhibitory interneurons show presymptomatic changes that may contribute to excitatory / inhibitory MN dysfunction in ALS.

Introduction

Amyotrophic lateral sclerosis (ALS) is a rapidly evolving adult-onset neurological disease characterized by a progressive loss of corticospinal neurons and MNs. There has been considerable debate in the field over the role of hyperexcitability in neurodegenerative processes in ALS. While drug treatment for ALS based on nonspecific reduction of neuronal excitability (with riluzole, for example) (Bellingham 2011), does result in a modest increase in lifespan (Bensimon et al., 1994), similar treatment paradigms have been disappointing clinically. A more nuanced understanding of the excitability of vulnerable neurons could help in creating a more targeted and effective approach for treatment of ALS. For example, if inhibitory pathways are failing in ALS and neuronal excitability is universally reduced with riluzole, this could further exacerbate inhibitory dysfunction by reducing activity not only in vulnerable MNs but also in inhibitory interneurons presynaptic to MNs. Thus it is important to consider all aspects of neuronal excitability, including intrinsic excitability of vulnerable neurons, synaptic drive, and neuromodulation (Gunes et al., 2020). Intrinsic properties of MNs have been well studied but the same is not true for interneurons that are synaptically connected to them. In fact, no studies thus far have directly assessed activity of spinal premotor interneurons in an ALS model.

Despite evidence that ALS patients have disrupted inhibition at spinal levels (Raynor and Shefner, 1994; Shefner and Logigian, 1998; Sangari et al., 2016; Howells et al., 2020; Özyurt et al., 2020), much is still unknown concerning the involvement of inhibitory circuitry in ALS. Morphological alterations in inhibitory circuits have been demonstrated in animal models of ALS. These include degeneration of spinal interneurons, fewer neurons expressing markers of inhibitory neurotransmitters including GlyT2 prior to loss of MNs (Martin et al., 2007; Hossaini et al., 2011), and loss of glycinergic boutons onto MNs prior to symptom onset (Chang and Martin, 2009a). Typically, fast MNs have greater numbers of inhibitory synaptic contacts, but these are largely lost in SOD1 mice beginning when motor unit atrophy is first observed (Pun et al., 2006; Hegedus et al., 2007; Allodi et al., 2021). Recurrent inhibitory circuits mediated by RCs are impaired before symptom onset by both loss of MN collaterals which provide synaptic drive, and complex changes to RC-MN synaptic structures (Casas et al., 2013; Wootz et al., 2013). A few studies have suggested activity is decreased in inhibitory interneurons by indirect measurements. Quantification of synaptic inputs to MNs has shown that frequency of inhibitory postsynaptic potentials in spinal MNs is decreased embryonically in both SOD1 mouse and zebrafish models (McGown et al., 2013; Branchereau et al., 2019) and glycinergic inputs to MNs decay faster in SOD1 MNs (Medelin et al., 2016). On a larger scale, blocking the activity of V1 inhibitory interneurons during in vivo treadmill running was recently found to mimic the early locomotor deficits in SOD1 mice (Allodi et al., 2021), suggesting that inhibitory interneurons could be inactive or under-active. However, none of these studies directly examined electrophysiological activity in inhibitory interneurons. We hypothesized that inhibitory spinal interneurons could contribute to the pathogenesis of ALS through a depression of MN inhibition. Decreased activity of inhibitory interneurons could result in synaptically-driven hyperexcitability of MNs and other long-term changes in network function. In this study, we examined electrical and morphological properties of glycinergic interneurons in the spinal cord of SODG93A GlyT2-eGFP (SOD1) mice compared to GlyT2-eGFP (WT) using whole cell patch clamp and three-dimensional reconstructions. We show here that significant dysfunction is present in SOD1 glycinergic interneurons which likely comprise several subclasses of inhibitory interneurons. In general, SOD1 glycinergic interneurons are less excitable and smaller than WT. Impairment in glycinergic interneurons should be explored as both a mechanism of vulnerability of MNs and a potential biomarker of early dysfunction of spinal circuits.

Results

Morphology of glycinergic interneurons

Ventrally located glycinergic interneurons in the lumbar enlargement of WT and SOD1 mice postnatal day (P) 6-10 were reconstructed from z-stack images obtained using confocal microscopy. SOD1 GlyT2 interneurons were significantly smaller than WT GlyT2 interneurons. Soma volume and surface area were both significantly smaller in SOD1 GlyT2 interneurons found throughout the ventral horn. Typical ventrolateral glycinergic neurons are shown in Figure 1. Complete reconstructions of soma morphology were performed on 4138 GlyT2 interneurons (2551 WT and 1587 SOD1 interneurons) from fixed lumbar spinal cords from 18 mice (9 WT and 9 SOD1). See Table 1 for all morphological parameters. Dendritic morphology was not possible in these images due to the high number of GFP+ neurons and processes but was completed on a smaller subset of neurons that were imaged live after patch clamp electrophysiology with a 2-photon microscope.

Figure 1:
Figure 1: GlyT2 interneurons in the ventral horn from SOD1 mice are smaller than WT.

Representative photomicographs of GFP interneurons in A. (B) Morphological analysis showed SOD1 glycinergic interneurons in ventral horn (N = 1587) were smaller in than WT (N = 2551). Significance denoted with *** indicates p ≤ 0.001. Scale bar in A: 100 μm applies to both images.

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Table 1:

Reconstruction data of GlyT2-GFP+ interneurons in WT and SOD1 animals.

To study electrophysiological properties of GlyT2 interneurons, 59 glycinergic neurons (34 WT and 25 SOD1) were recorded using visually guided patch clamp with Texas Red dye in the electrode as shown in Figure 2. The location of patched neurons was distributed throughout the ventral horn as shown in Fig 2B. In this smaller sample of neurons, soma sizes of patched SOD1 interneurons were not different (Fig 2C). However, dendrites were larger in patched SOD1 GlyT2 interneurons, including greater dendritic surface area and volume (see Table 1 for complete results). Please note that dimensions of these neurons cannot be directly compared to the previous section since these neurons were imaged in living tissue while the previous analysis was performed in fixed tissue, which shrinks.

Figure 2
Figure 2 GlyT2 interneurons from WT and SOD1 mice were patched throughout the ventral horn.

Images of typical interneurons, filled with Texas Red as they were recorded in A, and locations of all patched interneurons in B (WT = blue circles, SOD1 = red squares). (C) Soma volume and (D) soma surface area was unchanged in patched neurons, while dendrite surface area and volume were larger in SOD1 interneurons than in WT. Significance denoted with * indicates p ≤ 0.05.

Electrophysiology of glycinergic interneurons

Whole cell patch clamp revealed very different intrinsic properties of ventral glycinergic interneurons. Recording was performed on 59 ventral interneurons from throughout the ventral horn in transverse lumbar spinal cord slices from P6-10 mice. SOD1 inhibitory interneurons (n = 25) were found to have diminished intrinsic excitability compared to WT interneurons (n = 34). Measurements of intrinsic excitability included depolarized onset and peak voltage of PICs and depolarized threshold for action potential firing, as shown in Figure 3. In voltage clamp, slowly depolarizing voltage ramps were used to measure PICs (typical leak subtracted traces shown in Fig3A). Voltage sensitivity is determined by measuring voltage at PIC onset and peak. Both PIC onset and peak were significantly depolarized in SOD1 interneurons, while amplitude of the PIC was unchanged. Depolarizing current ramps were used to measure the input-output relationship of the neurons in current clamp. Inhibitory interneurons from SOD1 mice were found to have significantly higher threshold voltage than control interneurons (Fig 3E-F). This shift is likely driven by the changes in PIC. The current at firing onset, or I-ON, was not significantly changed (p = 0.15), similar to the current at firing offset, or I-OFF (p = 0.07). AHP was also significantly different in SOD1 interneurons. The duration (at half AHP amplitude) was shorter in SOD1 neurons, as shown in Fig 3H-I. Amplitude of the AHP was unchanged. While changes in the PIC and threshold indicate less excitability in SOD1 interneurons, the shortened AHP suggests an increased ability to fire at higher rates in SOD1 interneurons, a property that is associated with increased excitability. However, no changes in firing rates were detected in SOD1 interneurons. All other properties were found to be similar in SOD1 and WT glycinergic interneurons, including maximum firing rates, sag/rebound currents (IH), action potential parameters and membrane properties (see Table 2 for all electrical properties). See discussion for further interpretation of these findings.

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Table 2:

Electrophysiological properties of GlyT2-GFP+ interneurons in WT and SOD1 animals.

Figure 3:
Figure 3: Electrophysiology of SOD1 glycinergic interneurons.

The most prominent difference in SOD1 interneurons was the shift in voltage dependence of PICs and threshold. (A) Representative, leak-subtracted current-voltage relationship of PICs from WT (P8) and SOD1 (P6) interneurons. (B-D) Mean onset, peak and amplitude of PICs in WT (blue symbols on left) and SOD1 interneurons (red symbols on right). (E-F) Threshold voltage (action potentials evoked with current ramps as shown) was depolarized in SOD1 interneurons. Starting potential for both interneurons was −65mV. The current at firing onset (I-ON) was not significantly different as shown in G. (H) The AHP in SOD1 interneurons was shorter, as shown by the smaller mean duration at half amplitude in a representative P8 WT and P7 SOD1 interneuron and cumulative data in I. Neither the AHP amplitude nor the AHP decay time constant, tau, were significantly altered in SOD1 glycinergic interneurons, as shown in J-K. Vertical scale bars in E: top = 50pA, and bottom = 20mV. Horizontal scale bar in E = 0.5s and all scale bars apply to both left and right panels. Vertical scale bar in H = 10mV (APs were truncated from image), and horizontal scale bar = 50ms. Significance denoted with * indicates p ≤ 0.05; ** indicates p < 0.01.

Regional analysis of glycinergic interneurons

Since ventral glycinergic interneurons are not a homogenous population, we tested for regional differences. Interneurons were divided into three groups based on location 1) the “ventral RC region” within 100 μm of the ventral white matter where Renshaw cells are located, 2) lamina IX, and 3) the “intermediate region” of the ventral horn including lamina VII and VIII, excluding the previous two regions. These regions are shown in Figure 4. The first region corresponds to the location of RCs (but could contain other types of glycinergic interneurons). The second region contains interneurons that are interspersed with the MNs within the motor pools, likely to be composed of premotor interneurons (more on this in the discussion). The third region contains interneurons that are a mix of premotor interneurons and other classes of ventral interneurons. There was overlap of the first two regions, so interneurons within the ventralmost 100um that also were located within motor pools were included in both the ventral RC region and lamina IX, as shown in open circles in figure 4.

Figure 4:
Figure 4: Regional divisions of interneurons.

(A) Three regions were used for analysis of regional differences in glycinergic interneurons. (B) Map of the patched interneurons coded for regions. Ventral region with RC cells (green circles) overlapped with lamina IX (hollow circles). The remaining interneurons that were not within the RC region or lamina IX were grouped into an intermediate region of lamina VII and VIII (red triangles). Scale bar in A = 100 μm.

GlyT2 interneurons in Ventral RC region

Within the ventral region where RCs are located, we found that SOD1 interneurons there showed the most significant changes in both electrophysiology and morphology (particularly dendritic morphology). In patch clamped interneurons from this region (N = 8 WT from 7 mice and 13 SOD1 from 9 mice), there was a significant depolarization in PIC onset and peak in glycinergic SOD1 interneurons compared to WT interneurons in the same location. Additionally, dendrites were found to be longer and larger in volume and surface area in SOD1 interneurons in the ventral-most 100 μm. Unpatched GlyT2+ SOD1 interneurons in this region (129 WT interneurons from 8 WT mice and 168 SOD1 interneurons from 9 SOD1 mice) were smaller than WT. These changes (summarized in Figure 5 and Table 3) show that glycinergic neurons in the region where RCs are located are very affected by the SOD1 mutation.

Figure 5:
Figure 5: PICs were depolarized in SOD1 GlyT2 interneurons in the ventral RC region.

Both onset (A) and peak (B) of the PIC were depolarized, but voltage threshold (C) was not significant. Soma volume of patched neurons (D) was unchanged, though dendrites of SOD1 interneurons had more volume (E) and were longer (F). Larger numbers of unpatched neurons showed smaller soma volume (G) and surface area (H) in SOD1 interneurons than WT, in RC region. Significance denoted with * indicates p ≤ 0.05, ** indicates p ≤ 0.01.

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Table 3:

Electrophysiological properties of GlyT2-GFP+ interneurons in WT and SOD1 animals in ventral RC region.

GlyT2 interneurons in Lamina IX

The SOD1 interneurons located in lamina IX, intermixed with MNs were modestly altered in excitability and morphology. As shown in Figure 6 and Table 4, PIC onset was depolarized in SOD1 interneurons in this region, compared to interneurons in this region from WT mice (n = 14 WT interneurons from 8 mice and 11 SOD1 interneurons from 9 mice). Longer dendritic length was also found in patched interneurons in this region, and within the larger number of unpatched interneurons, (345 WT interneurons from 7 WT mice and 306 SOD1 interneurons from 8 SOD1 mice) SOD1 interneurons were found to have smaller somata than WT interneurons.

Figure 6:
Figure 6: Electrophysiology and morphology was altered in glycinergic SOD1 interneurons in lamina IX.

PIC onset (A), PIC peak (B) and voltage threshold (C) were all depolarized in SOD1 interneurons. Soma (D) and dendrites (E) and (F) of patched SOD1 interneurons were unchanged. Larger numbers of unpatched neurons showed smaller soma volume (G) and surface area (H) in SOD1 interneurons than WT in Lamina IX. Significance denoted with * indicates p ≤ 0.05, *** indicates p ≤ 0.001.

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Table 4:

Electrophysiological properties of GlyT2-GFP+ interneurons in WT and SOD1 animals in lamina IX.

GlyT2 interneurons in the Intermediate region

The third regional group of glycinergic interneurons was defined as the intermediate region. These interneurons were located in laminae VII and VIII, excluding the ventral 100 μm (and of course excluding lamina IX). Within this area, 16 WT and 8 SOD1 interneurons were patched from 11 WT and 5 SOD1 mice respectively, and 2077 unpatched interneurons were analyzed from 9 WT mice and 1113 unpatched SOD1 interneurons from 9 SOD1 mice. As shown in Figure 7 and Table 5, we found little was changed in electrophysiological parameters of SOD1 interneurons (only PIC peak was significantly depolarized). In unpatched neurons from this region, we found that soma size was significantly smaller in SOD1 interneurons, similar to the other two regions.

Figure 7:
Figure 7: Morphology was altered in SOD1 interneurons in the intermediate regions of laminae VII and VIII.

PIC onset (A) was not significantly different in SOD1 interneurons, though PIC peak (B) was depolarized. Voltage threshold (C) was not affected in SOD1 interneurons. Morphology of soma (D) and dendrites (E) in patched SOD1 interneurons were unchanged, however larger numbers of unpatched neurons showed smaller soma volume (G) and surface area (H) in SOD1 interneurons than in WT, in laminas VII and VIII. Significance denoted with * indicates p ≤ 0.05, *** indicates p ≤ 0.001.

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Table 5:

Electrophysiological properties of GlyT2-GFP+ interneurons in WT and SOD1 animals in Intermediate Region of lamina VII and VIII.

Taken together, these results show that glycinergic interneurons are profoundly affected by the SOD1 mutation and are altered very early in life in both electrophysiology and morphology. These changes are not universal to all glycinergic interneurons, but rather are specific to various subtypes of inhibitory interneurons.

Discussion

This study shows for the first time that glycinergic interneurons are depressed in excitability in the SOD1 mouse model and may be contributing to ALS pathology. Alterations in both morphology and excitability of glycinergic interneurons occur at a very early stage. GlyT2 interneurons in SOD1 mice are smaller and depressed in excitability, particularly those within 100um of the ventral white matter, as summarized in Figure 8. We speculate that these shifts in interneuron excitability and morphology could have functional consequences that alter inhibition of MNs, modify motor output, and may even provide an early biomarker of ALS.

Figure 8:
Figure 8:

SOD1 GlyT2 interneurons are most profoundly altered in the ventral-most 100μm of the spinal cord, the area where Renshaw cells are found. All ventral interneurons showed changes in morphology and excitability, including depolarization of PICs and smaller somata. Interneurons in the ventral region and lamina IX showed increased dendritic length. Interneurons in the ventral region had the most strongly affected electrophysiology (both PIC onset and peak depolarized).

Intrinsic and synaptic hyperexcitability

A core issue this study brings to light is potential long-term disruption to the balance of excitatory and inhibitory synaptic transmission in ALS. Spinal cord and cortex both show an imbalance between excitatory and inhibitory synaptic transmission in SODG93A mice (Avossa et al., 2006; Saba et al., 2016). Clinically, ALS patients show evidence of altered synaptic activity as well, including a reduction in inhibition. A reduction of glycinergic receptor binding in the anterior gray matter of the spinal cord (Hayashi et al., 1981; Whitehouse et al., 1983) has been demonstrated along with abnormal glycine and gamma amino butyric acid (GABA) levels in blood serum (Malessa et al., 1991; Niebroj-Dobosz and Janik, 1999). Electrophysiology also suggests disruption in spinal inhibitory circuits in ALS patients (Raynor and Shefner, 1994; Shefner and Logigian, 1998; Sangari et al., 2016; Özyurt et al., 2020). An interesting question is whether the neurons are altered in excitability only within the motor circuit (i.e. corticospinal neurons and the synaptically-connected spinal inhibitory and cholinergic interneurons, and MNs), or if disturbances are more widespread throughout the nervous system. In the spinal cord, interneurons that serve as conduits between vulnerable corticospinal and spinal MNs include cholinergic interneurons, Ia inhibitory interneurons and RCs. There is abundant evidence of disruption in these cholinergic, GABAergic and glycinergic synapses from animal models (Martin et al., 2007; Chang and Martin, 2009b; Pullen and Athanasiou, 2009; Hossaini et al., 2011; Herron and Miles, 2012; Casas et al., 2013; Wootz et al., 2013; Saxena et al., 2013; Milan et al., 2015; Dukkipati et al., 2016; Medelin et al., 2016). Similarly, in the cortex, neurons that are not themselves vulnerable to neurodegeneration in ALS also exhibit altered excitability and morphology in a parallel time course to the vulnerable corticospinal neurons (Clark et al., 2017, 2018; Kim et al., 2017). The changes in excitability of other neuronal populations must contribute to the imbalance of both excitatory and inhibitory neurotransmission that has been clearly demonstrated in ALS patients and animal models.

A case for hyperexcitability and glutamate excitotoxicity involvement in ALS pathogenesis has been more controversial. Altered intrinsic excitability has been consistently reported in both corticospinal projection neurons and spinal MNs, but these perturbations appear to fluctuate based on age/disease progression. Corticospinal projection neurons have increased firing and other signs of hyperexcitability during the first week of postnatal development (Pieri et al., 2009; Saba et al., 2016, 2019; Kim et al., 2017), but excitability is normal in young and adult pre-symptomatic ages (Kim et al., 2017; Saba et al., 2019), and may become hypoexcitable at the age of symptom onset (Saba et al., 2019). Similarly, spinal MNs show intrinsic hyperexcitability at very early (embryonic) stages (Kuo et al., 2004, 2005; Martin et al., 2013). At a postnatal age similar to the inhibitory interneurons in this study, spinal MNs do not have an altered threshold, rheobase or frequency-current relationship, though other abnormalities are present in PICs, action potential duration, AHP and dendritic Ca2+ entry (Pambo-Pambo et al., 2009; Quinlan et al., 2011, 2015; Leroy et al., 2014). Thus, at the age studied here, inhibitory interneurons are more disrupted in excitability than the vulnerable spinal MNs. At symptom onset, MNs may finally succumb to hypoexcitability, though not all studies agree on this point (Delestrée et al., 2014; Martinez-Silva et al., 2018; Jensen et al., 2020). Notably, MNs derived from ALS patients’ induced pluripotent stem cells also show initial hyperexcitability followed by hypoexcitability (Wainger et al., 2014; Devlin et al., 2015), and increasing excitability of MNs enhanced neuroprotection rather than neurodegeneration (Saxena et al., 2013). A unifying theme throughout the findings is that vulnerable MNs show excessive homeostatic gain in response to perturbation (Kuo et al., 2020), and not only excitability but other cellular properties fluctuate over the lifespan of the animal (Irvin et al., 2015). In light of that, whether inhibitory interneurons are less excitable at other ages should be explored in future studies to fully characterize their contribution to neurodegenerative processes.

Changes in neuron morphology

In ALS models, altered morphology in spinal MNs occurs so early that it could be viewed as a deficit in normal development. Embryonically, SOD1 MNs have shorter projections but this is reversed during postnatal development. At 1-2 weeks of age, MNs begin to show more dendritic branching and larger soma sizes (Amendola and Durand, 2008; Filipchuk and Durand, 2012; Martin et al., 2013). Interestingly, this could be caused by lack of inhibitory input to MNs (Fogarty et al., 2016). Spinal MNs remain larger through adulthood (Dukkipati et al., 2018). In contrast to the spinal MNs at this age, we found that inhibitory interneurons had smaller soma size and yet had expanded dendritic surface area and volume. This is reminiscent of changes in corticospinal neurons: presymptomatic corticospinal neurons also have increased arborization but with smaller soma diameters (Ozdinler et al., 2011; Saba et al., 2016). In both sporadic and familial ALS patients, postmortem tissue shows degeneration of apical dendrites in corticospinal neurons and smaller soma size (Genç et al., 2017). In the spinal interneurons, increased dendrite length, surface area and volume occurs in specific populations of interneurons: the increased dendritic arbor is specifically found in the ventral interneurons, while the smaller soma sizes are more universal. Increased neuronal size / arborization over the life span could greatly increase the impact of other stressors (e.g., proteostasis, metabolic deficits, intracellular transport) on vulnerable neuron populations.

After spike afterhyperpolarization

The AHP can contribute to a neuron’s firing rate such that a neuron with a large, long-lasting AHP will typically fire at a slower rate (typical of slow/type I MNs) than a neuron with small, fast-decaying AHP (typical of fast/type II MNs). In ALS patients, the AHP is shortened in MNs of patients with little force deficits and later elongated in patients with large force deficits (Piotrkiewicz and Hausmanowa-Petrusewicz, 2011), which could reflect early changes in physiology of vulnerable fast MNs and later, the remaining, less-vulnerable population of slow MNs. Similarly, SOD1 mouse MNs show shorter AHPs early, and longer AHPs around symptom onset (Quinlan et al., 2011; Jensen et al., 2020). Here we show that AHP is shorter in duration in SOD1 interneurons very early in life. In the regional analysis, AHP parameters were not found to be significantly altered in any of the 3 regions, but it is possible this was due to a loss in statistical power in the smaller groups. We did not find concomitant changes in minimum or maximum firing rates in GlyT2 interneurons, so the physiological importance of the altered AHP in interneurons is unclear.

Inhibitory interneurons: Subtype specific effects

The interneurons in this study were heterogenous, and based on their location and electrophysiology, they likely were composed of V1 and V2b interneurons including RCs, commissural interneurons (V2b interneurons), and Ia inhibitory interneurons, and perhaps inhibitory propriospinal interneurons (Willis and Willis, 1964; Restrepo et al., 2009; Bikoff et al., 2016; Flynn et al., 2017). Because of this heterogeneity, interneurons were separated into regions for further analysis, to examine more specifically which inhibitory interneurons were perturbed. Based on our regional analysis, interneurons in the ventral-most 100um were altered the most in excitability, with the most strongly depolarized PICs, moreso than the glycinergic interneurons within lamina IX and intermediate lamina VI and VII. Interneurons located in the region within 100μm from the ventral white matter likely contain RCs based on the location (Siembab et al., 2010) and electrophysiological characteristics (Perry et al., 2015; Bikoff et al., 2016). Glycinergic interneurons located in lamina IX are reminiscent of the locomotor-related GABAergic interneurons characterized by Nishimaru and colleagues, which were located within the ventrolateral spinal cord, surrounded MNs and were active during locomotor activity (Nishimaru et al., 2011). Indeed these may be the same neurons: colocalization of GABA and glycine is entirely possible (Jonas et al., 1998; Allain et al., 2006). If the lamina IX interneurons are indeed locomotor-related, even a modest dampening of excitability observed here in SOD1 mice could contribute to changes in locomotor patterning as has been described in other studies (Quinlan et al., 2017; Allodi et al., 2021). Further studies are warranted to examine how inhibitory interneurons may be differently modulated in a subtype-specific manner in animal models of ALS.

Functional implications for impaired inhibitory circuits

Outward signs of improperly functioning inhibitory interneurons may be apparent from subtle changes in motor patterning and could potentially be used as a biomarker of early ALS. Locomotor disturbances have been demonstrated in vivo in presymptomatic SOD1 mice during walking/running (Vinsant et al., 2013a, 2013b; Akay, 2014; Quinlan et al., 2017; Allodi et al., 2021). In the embryonic and neonatal spinal cord in vitro, increased duration of depolarizing events in MNs and a slower period of locomotor-related bursting has been described (Medelin et al., 2016; Branchereau et al., 2019), which indicate that even during development the functioning of locomotor circuits is already impaired. In presymptomatic adult mice capable of running on a treadmill, subtle locomotor differences are observed including advanced intermuscular phasing and a slower speed (Quinlan et al., 2017; Allodi et al., 2021) which indicate that before any large scale neurodegeneration occurs, there are changes in patterns of activity in MNs and interneurons that could be exploited for use as a biomarker of ALS. At initial symptom onset, the first failure of the tibialis anterior manifests as abnormality in the swing phase of the ankle (Akay, 2014). Interestingly, silencing different interneuron populations (including inhibitory interneurons and cholinergic interneurons) reveals the contribution of each population: silencing cholinergic interneurons increases locomotor deficits in SOD1 mice (Landoni et al., 2019), while silencing inhibitory interneurons has no effect suggesting these interneurons are already impaired (Allodi et al., 2021).

Clinical-translational implications

Evidence of MN hyperexcitability in ALS patients is present alongside signs of dysfunction in inhibition (Rothstein et al., 1992; Plaitakis and Constantakakis, 1993; Raynor and Shefner, 1994; Shefner and Logigian, 1998; Andreadou et al., 2008; Bae et al., 2013). Perhaps reducing excitability (with riluzole, for example) only results in a modest increase in lifespan (Bensimon et al., 1994) because it could be helpful to MNs while exacerbating inhibitory dysfunction by further depressing excitability of inhibitory interneurons like RCs. Thus, a more targeted approach to reduce MN excitability could be beneficial early in disease pathology. Stimulation of inhibitory nerves or certain protocols of transcranial stimulation could be explored, along with augmentation of inhibitory neurotransmission.

Materials and Methods

Ethics Statement

Experiments were performed in accordance with the United States National Institutes of Health Guide for Care and Use of Laboratory Animals. Approval of the Northwestern University’s Animal Care and Use Committee was obtained for all experiments performed in this study. All efforts were made to minimize animal suffering and to reduce the number of animals used.

Animals and Tissue harvest

Transgenic B6SJL mice overexpressing the human SOD1G93A gene (strain 002726, Jackson Labs, Bar Harbor, ME, USA) and their wild type littermates were used (nontransgenic for the human SOD1G93A gene). Transgenic animals were identified using standard PCR techniques by Transnetyx (Cordova, TN, USA) and were bred with GlyT2-eGFP mice (Zeilhofer et al., 2005) generating SOD1G93A GlyT2-eGFP mice, here called SOD1. Inhibitory glycinergic interneurons express the Na+ and Cl- coupled glycine transporter 2, or GlyT2. GlyT2-eGFP expression is driven by the GlyT2 promotor. Specifically, GlyT2-eGFP females were bred with SOD1G93A GlyT2-eGFP males, and progeny were used for experiments without knowing if they were SOD1G93A GlyT2-eGFP or GlyT2-eGFP. Genotyping was performed post-experimentally. For the following studies juvenile mouse pups were used between postnatal day (P) 6 - 10. Mice were deeply anesthetized with isoflurane (Henry Schein Animal Health, Dublin, OH, USA), decapitated and eviscerated. The lumbar spinal cord from L1 - L6 was removed and embedded in 2.5% w/v agar (No. A-7002, Sigma-Aldrich, St Louis, MO, USA). The agar block was then superglued with Loctite 401 (Henkel Corporation, Rocky Hill, CN, USA) to a stainless steel slicing chuck and 350 μm transverse slices were made using the Leica 1000 vibratome (Leica Microsystems, Buffalo Grove, IL, USA) as described previously (Quinlan et al., 2011). During both spinal cord isolation and slicing, the spinal cord was immersed in 1–4°C high osmolarity dissecting solution containing (mM): sucrose 234.0, KCl 2.5, CaCl2 · 2H2O 0.1, MgSO4 · 7H2O 4.0, HEPES 15.0, glucose 11.0, and Na2PO4 1.0. The pH was adjusted to 7.35 when bubbled with 95% O2/5% CO2 using 1 M KOH (Fluka Analytical, Sigma-Aldrich). After cutting, the slices were incubated for >1 h at 30°C in incubating solution containing (mM): NaCl 126.0, KCl 2.5, CaCl2 · 2H2O 2.0, MgCl2 · 6H2O 2.0, NaHCO3 26.0, glucose 10.0, pH 7.4 when bubbled with 95% O2/5% CO2 (all reagents for solutions were purchased from Sigma-Aldrich).

Morphological analysis

Neuron morphology was assessed from two types of images: 1) 2-photon image z-stacks of live neurons taken immediately after obtaining electrophysiological parameters, and 2) confocal image stacks taken after fixation and tissue processing. Images collected using these two methods were analyzed separately. Measurements from 2-photon images included measurements from both soma and dendrites (soma volume, soma surface area, and average dendrite length, dendrite surface area and dendrite volume). Data from confocal images only included soma measurements, specifically soma volume and soma surface area. The reconstruction is made based on the sum of many sections of image (z stacks), resulting in the 3D image. Volume is computed by sum of each section area, multiplied by the thickness, resulting in the volume; the sum of each section perimeter of the reconstruction multiplied by the thickness, resulting in the surface; dendrites analysis, computed by modeling as the previous ones using each piece of each branch as a frustum.

Two-photon imaging

An Olympus BX-51WIF microscope fitted with an Olympus 40x/0.8NA water-dipping objective lens was used. Two-photon excitation fluorescence microscopy was performed with a galvanometer-based Coherent Chameleon Ultra II laser (Coherent, Santa Clara, CA, USA) tuned to 900 nm. A red Bio-Rad 2100MPD photomultiplier tube (Bio-Rad, Hercules, CA, USA) (570 – 650 nm) was used to collect emission in the red channel. Z-stacks were obtained for each interneuron at 1024 x 1024 pixels (308 x 308 μm) resolution and roughly 100 μm in depth (step size 1 μm). From these Z-stacks, Texas Red®-filled neurons were three-dimensionally reconstructed using Neurolucida software (MBF Bioscience, Williston, VT, USA).

Confocal imaging

Sections were observed with a confocal laser microscope (LSM510, Zeiss, Oberkochen, Darmstadt, Germany) with argon (488 nm) and helium-neon (543 nm) lasers and a 20X objective. From these Z-stacks, GlyT2 positive interneurons were three-dimensionally reconstructed using Neurolucida software (MBF Bioscience, Williston, VT, USA). Dimensions of neurons in each image stack were averaged before compiling numbers. Total numbers of analyzed interneurons was 3,902 composed of 1918 neurons from 36 WT image stacks and 1984 interneurons from 44 SOD1 image stacks.

Electrophysiology

Whole cell patch clamp was performed on MNs from the lumbar segments using 2–4 MΩ glass electrodes pulled from glass capillary tubes (Item #TW150F-4, World Precision Instruments, Sarasota, FL, USA) with a Flaming-Brown P-97 (Sutter Instrument Company, Novato, CA, USA). Electrodes were positioned using a Sutter Instrument MP-285 motorized micromanipulator (Sutter Instrument Company). Whole-cell patch clamp measurements were performed at room temperature using the Multiclamp700B amplifier (Molecular Devices, Burlingame, CA, USA) and Winfluor software (University of Strathclyde, Glasgow, Scotland). Briefly, slices were perfused with a modified Ringer’s solution containing (in mM): 111 NaCl, 3.09 KCl, 25.0 NaHCO3, 1.10 KH2PO4, 1.26 MgSO4, 2.52 CaCl2, and 11.1 glucose. The solution was oxygenated with 95% O2/5% CO2 and the perfusion rate was 2.5 – 3.0 ml/min. Patch electrodes contained (in mM) 138 K-gluconate, 10 HEPES, 5 ATP-Mg, 0.3 GTP-Li and Texas Red dextran (150 μM, 3000 MW, from Invitrogen, Life Technologies, Grand Island, NY, USA). In voltage-clamp mode, fast and slow capacitance transients, as well as whole-cell capacitance, were compensated using the automatic capacitance compensation on the Multiclamp. Whole cell capacitance was recorded from the Multiclamp.

Electrophysiological analysis

Holding potential was set at −90 mV, and neurons were subjected to slow, depolarizing voltage ramps of 22.5 mV s−1, bringing the cell to 0 mV in 4 s, and then back to the holding potential in the following 4 s. In current clamp, neurons were subjected to depolarizing current ramps for testing I-on (the current level at firing onset), I-off (the current level at cessation of firing), and the slope of the frequency–current relationship. The difference between I-on and I-off is calculated for ΔI. Negative current was often necessary to prevent action potential (AP) firing. Resting membrane potential was recorded as the potential when 0 current was injected. Persistent inward current (PIC) parameters were measured after subtraction of leak current. Onset was defined as the voltage at which the current began to deviate from the horizontal, leak-subtracted trace. PIC peak was the voltage at which the PIC reached peak amplitude. The linear portion of leak current (usually between −90 and −75 mV) was used to calculate whole cell input resistance. The first action potential in the train evoked by a depolarizing current ramp in current clamp mode was used to measure all parameters relating to action potentials. Threshold voltage was defined as the voltage at which the slope exceeded 10 V/s. Threshold for action potential firing was tested in two ways. The first was to use the voltage at firing onset from the current ramps up to 130 pA/s (in current clamp). The second was the voltage at which a single action potential could be evoked with a current step. Action potential overshoot is the voltage past 0 mV the first spike reaches. Duration of the action potential is measured at half of action potential height (height is defined as overshoot – threshold voltage). Rates of rise and fall are defined as the peak and the trough of the first derivative of the action potential profile. Instantaneous firing frequency range max was the maximum firing rate that could be evoked from a neuron. It was measured from the first or second firing frequencies on a large (>150pA) depolarizing step. Steady state firing freq range minimum was the lowest firing frequency evoked from a small (~50pA) depolarizing step. Hyperpolarizing steps were used to measure IH. The sag and rebound were measured from largest evoked response. Sag potential was calculated as a % of the total amplitude of the hyperpolarization. If a spike was evoked on rebound, the voltage threshold was used for measurement. AHP duration was measured from a single spike fired during a period in which the membrane potential was stable. Amplitude was measured as the downward deflection from the membrane potential. Duration of the AHP was measured from the falling phase of the spike at baseline potential to the time of recovery to baseline membrane potential. Half amplitude duration was measured as the duration at half of the measured AHP amplitude from the falling phase of the spike at baseline potential. Tau is measured as the rate of decay from the last third of the AHP.

Neuron Selection

Glycinergic interneurons were visually selected for recording based on 1) expression of GFP, and 2) location near the MN pools. Neurons that did not repetitively fire or did not maintain a resting membrane potential below −35 mV were excluded from electrophysiological analysis. Neurons that were not clearly stained with Texas Red after patch clamp and/or could not be imaged clearly were not included in anatomical analysis.

Regional grouping

Photos were taken of position of the patch electrode within the spinal cord slices after recording was complete. These photos were used to recreate a map of the position of the interneurons. Interneurons within the ventralmost region of the spinal cord (within 100μm of the ventral white matter) were grouped into the RC region described previously (Siembab et al., 2010). Neurons that were observed to be located within and on the lateral edges of the motor pools (in between motoneuron and the white matter) were classified as lamina IX interneurons. Since the location of the motor pools varied throughout the lumbar enlargement, this classification was determined by examination of the z stacks and photos. Neurons that were not found within lamina IX and were not within the ventral RC region were grouped as the intermediate region of Lamina VII/VIII.

Statistical analysis

The assumptions of homogeneity of variances and the normality of the distribution of values for each measured characteristic were evaluated with Levine’s and Shapiro-Wilk tests, respectively. Group comparisons (WT vs SOD1) were performed using one-way ANOVA on parameters that satisfied both conditions (homogeneity and normality of distribution) and using the Kruskal-Wallis test for those that did not. Results are presented as means +/−SEM. Significance level was set at p≤ 0.05.

Acknowledgments

This project was funded by a springboard fellowship from Target ALS to KAQ and a University of Rhode Island Graduate School Tuition Scholarship to LMM. The authors made use of equipment supported by the Institutional Development Award (IDeA) Network for Biomedical Research Excellence from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103430. The authors thank Drs Emily Reedich, Claudia Fallini and Marin Manuel for comments on the initial drafts of the manuscript.

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Posted June 22, 2021.
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Inhibitory interneurons show early dysfunction in a SOD1 mouse model of ALS
C. F. Cavarsan, P. R. Steele, L. M. McCane, K. J. LaPre, A. C. Puritz, N. Katenka, K. A. Quinlan
bioRxiv 2020.10.21.348359; doi: https://doi.org/10.1101/2020.10.21.348359
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