**3.2 The changes in action potential and afterhyperpolarization**

A change in the action potential properties was frequently seen in mutant motoneurons relative to WT; however, these changes were conflicting across the transgenic models (Table 1, Fig. 2). In the G93A (high expressor line) model, neonatal spinal motoneurons frequently showed faster action potential rate of rise and decay and shorter action potential duration (Pieri et al., 2003; Quinlan et al., 2011), indicating an increase in transient and persistent Na+ currents, which act to increase the excitability of mutant motoneurons. Signs of increased excitability were differently displayed in other studies as an increase in spike height (van Zundert et al., 2008), depolarization in resting membrane potential (Kuo et al., 2005), or reduction in action potential threshold (Pieri et al., 2009). Conversely, an increase in action potential duration and deceleration in rate of repolarization were observed in postsymptomatic adult hypoglossal motoneurons (Fuchs et al., 2009).

Electrophysiological Abnormalities in SOD1 Transgenic

increased only in the G93A (high expressor line) transgenic model.

Fig. 3. The changes in ramp frequency-current relationship gain (ramp F-Igain), sodium (NaPIC) and calcium (CaPIC) persistent inward currents in mutant motoneurons of the G93A (high expressor line) [2nd bars]1, G93A (high expressor line) [3rd bars]2, and the G85R

[last bars]2 transgenic models, relative to WT [1st bars]. All reported data from slice

A change in the motoneuron gain (i.e., F-I relationship slope) or firing activity was commonly seen in mutant motoneurons, but conflicting observations were also reported. In the G93A (high expressor line) transgenic model, the firing activity and gain of mutant spinal motoneurons were consistently increased in cell culture (Pieri et al., 2003; Kuo et al.,

preparation. 1: Quinlan et al. (2011), 2: Pambo-Pambo et al. (2009).

**3.4 The change in motoneuron gain and firing activity** 

Models in Amyotrophic Lateral Sclerosis: The Commonalities and Differences 165

measurement of the persistent inward current, whereas the ΔI technique (which is obtained from the difference in injected current at motoneuron recruitment and derecruitment on a triangular current command) provides an indirect estimate of the persistent inward current amplitude (Bennett et al., 2001) and is sensitive to the inactivation of transient Na+ channels (Miles et al., 2005) and activation of residual outward currents present in the motoneuron membrane (Hamm et al., 2010). However, it should be noted that the number of cells in which the voltage-clamp measurements were obtained was small. In the infrequently studied G127X transgenic model, an increase in the persistent inward current amplitude was estimated from the motoneuron firing profile (Meehan et al., 2010). However, this result is unconfirmed given that firing profile in mutant motoneurons would be also influenced by any differences in inward or outward currents and electrical properties of mutant motoneurons. Accordingly, the change in amplitude of persistent inward currents is not a common abnormality in the various ALS transgenic models. However, it appears that persistent inward currents are

Fig. 2. The changes in action potential height (APamp), maximum rise (APmax rise) and fall (APmax fall) rates, and AHP amplitude (AHPamp) and half-width (AHPhalf-width) in mutant motoneurons of the G93A (high expressor line) [2nd bars]1, G93A (high expressor line) [3rd bars]2, and the G85R [last bars]2 transgenic models, relative to WT [1st bars]. All reported data from slice preparation. 1: Quinlan et al. (2011), 2: Pambo-Pambo et al. (2009).

In the G93A (low expressor line) model, neonatal spinal motoneurons exhibited a reduction in action potential height, increase in half-width, and deceleration in the rates of rise and decay (Pambo-Pambo et al., 2009; Filipchuk et al., 2010). In the G85R model, no changes were observed in the properties of the action potential regardless of the tissue preparation (whole-cord or slice) and extracellular Ca2+ concentration (2mM or 4mM) (Bories et al., 2007; Pambo-Pambo et al., 2009; ElBasiouny et al., 2010). Therefore, it appears that the alteration in action potential properties is not a common pathological characteristic, but depends on the ALS transgenic model type.

The after-spike afterhyperpolarization was not consistently altered in mutant motoneurons across the various transgenic models (Table 1, Fig. 2).

### **3.3 The change in persistent inward currents**

Persistent inward currents are intrinsic Na+ and Ca2+ currents that depolarize the membrane potential when activated. Contrary to transient ionic currents, these currents do not inactivate with prolonged membrane depolarization (Schwindt and Crill, 1977). A change in the motoneuron persistent inward current was frequently observed in mutant motoneurons (Table 1, Fig. 3); however, conflicting data were reported on the nature of this change (i.e., increase or decrease). In the G93A (high expressor line) model, an increase in the Na+ persistent inward current of neonatal mutant motoneurons was consistently reported regardless of tissue preparation (cell culture and slice) or extracellular Ca2+ concentration (Table 1). In the G93A (low expressor line) and G85R models, persistent inward current measured in voltage-clamp protocols indicated no change in their amplitude between WT and mutant motoneurons, whereas persistent inward current estimated from the delta I (ΔI) technique indicated a reduction in their amplitude in mutant motoneurons relative to WT (Pambo-Pambo et al., 2009). The former is a more truthful result because voltage-clamp protocols allow direct

Fig. 2. The changes in action potential height (APamp), maximum rise (APmax rise) and fall (APmax fall) rates, and AHP amplitude (AHPamp) and half-width (AHPhalf-width) in mutant motoneurons of the G93A (high expressor line) [2nd bars]1, G93A (high expressor line) [3rd bars]2, and the G85R [last bars]2 transgenic models, relative to WT [1st bars]. All reported

In the G93A (low expressor line) model, neonatal spinal motoneurons exhibited a reduction in action potential height, increase in half-width, and deceleration in the rates of rise and decay (Pambo-Pambo et al., 2009; Filipchuk et al., 2010). In the G85R model, no changes were observed in the properties of the action potential regardless of the tissue preparation (whole-cord or slice) and extracellular Ca2+ concentration (2mM or 4mM) (Bories et al., 2007; Pambo-Pambo et al., 2009; ElBasiouny et al., 2010). Therefore, it appears that the alteration in action potential properties is not a common pathological characteristic, but depends on the

The after-spike afterhyperpolarization was not consistently altered in mutant motoneurons

Persistent inward currents are intrinsic Na+ and Ca2+ currents that depolarize the membrane potential when activated. Contrary to transient ionic currents, these currents do not inactivate with prolonged membrane depolarization (Schwindt and Crill, 1977). A change in the motoneuron persistent inward current was frequently observed in mutant motoneurons (Table 1, Fig. 3); however, conflicting data were reported on the nature of this change (i.e., increase or decrease). In the G93A (high expressor line) model, an increase in the Na+ persistent inward current of neonatal mutant motoneurons was consistently reported regardless of tissue preparation (cell culture and slice) or extracellular Ca2+ concentration (Table 1). In the G93A (low expressor line) and G85R models, persistent inward current measured in voltage-clamp protocols indicated no change in their amplitude between WT and mutant motoneurons, whereas persistent inward current estimated from the delta I (ΔI) technique indicated a reduction in their amplitude in mutant motoneurons relative to WT (Pambo-Pambo et al., 2009). The former is a more truthful result because voltage-clamp protocols allow direct

data from slice preparation. 1: Quinlan et al. (2011), 2: Pambo-Pambo et al. (2009).

ALS transgenic model type.

across the various transgenic models (Table 1, Fig. 2).

**3.3 The change in persistent inward currents** 

measurement of the persistent inward current, whereas the ΔI technique (which is obtained from the difference in injected current at motoneuron recruitment and derecruitment on a triangular current command) provides an indirect estimate of the persistent inward current amplitude (Bennett et al., 2001) and is sensitive to the inactivation of transient Na+ channels (Miles et al., 2005) and activation of residual outward currents present in the motoneuron membrane (Hamm et al., 2010). However, it should be noted that the number of cells in which the voltage-clamp measurements were obtained was small. In the infrequently studied G127X transgenic model, an increase in the persistent inward current amplitude was estimated from the motoneuron firing profile (Meehan et al., 2010). However, this result is unconfirmed given that firing profile in mutant motoneurons would be also influenced by any differences in inward or outward currents and electrical properties of mutant motoneurons. Accordingly, the change in amplitude of persistent inward currents is not a common abnormality in the various ALS transgenic models. However, it appears that persistent inward currents are increased only in the G93A (high expressor line) transgenic model.

Fig. 3. The changes in ramp frequency-current relationship gain (ramp F-Igain), sodium (NaPIC) and calcium (CaPIC) persistent inward currents in mutant motoneurons of the G93A (high expressor line) [2nd bars]1, G93A (high expressor line) [3rd bars]2, and the G85R [last bars]2 transgenic models, relative to WT [1st bars]. All reported data from slice preparation. 1: Quinlan et al. (2011), 2: Pambo-Pambo et al. (2009).

### **3.4 The change in motoneuron gain and firing activity**

A change in the motoneuron gain (i.e., F-I relationship slope) or firing activity was commonly seen in mutant motoneurons, but conflicting observations were also reported. In the G93A (high expressor line) transgenic model, the firing activity and gain of mutant spinal motoneurons were consistently increased in cell culture (Pieri et al., 2003; Kuo et al.,

Electrophysiological Abnormalities in SOD1 Transgenic

**Model MN** 

(high) ↑↑ ↓↓

(low) ↑↑ --

G93A

G93A

Models in Amyotrophic Lateral Sclerosis: The Commonalities and Differences 167

G85R ↑↑ ↓↓ -- -- ↓↓ Hypoexcitability

In the G85R transgenic model, not many changes were reported in the electrical properties of mutant motoneurons; however, these changes were consistent with reduced excitability and are supported by the decrease in gain of mutant motoneurons (Table 3). In conclusion, motoneuron pathophysiology is different in the various transgenic mouse models of ALS and their excitability varies from hypo- to hyperexcitability. This disparity could be related to the number of copies of the human SOD1 gene or the severity of the disease (transgenic models with high copy number of SOD1 genes have more aggressive ALS than transgenic models with low copy number of SOD1 genes). Transgenic models with a low number of SOD1 copies (e.g., G85R and G93A-low expressor line) showed a tendency for reduced excitability of mutant motoneurons, whereas transgenic models with a high number of SOD1 copies (e.g., G93A-high expressor line) showed a tendency for increased excitability of mutant motoneurons. These differences should be considered when studying the

Table 3. Summary of changes in motoneuron properties in the various ALS transgenic models. MN: motoneuron, Rin: input resistance, AP/AHP: action potential and

**4. Effect of anatomical alterations on motoneuron electrical properties** 

The enlarged anatomy of mutant motoneurons has consequences for their electrical properties and firing behaviour. Computer simulations have been used to assess the effect of enlargement in motoneuron anatomy and its contribution to the changes in electrical properties (ElBasiouny et al., 2010). Realistic computer models were developed from the reconstructed morphologies of WT and mutant motoneurons of the G85R model and were optimized to replicate the electrophysiological recordings obtained from individual cells. For the 30% reduction in input resistance of mutant motoneurons relative to WT, computer simulations showed that one third of this reduction (i.e., input resistance decrease by 10%) is due to the enlargement of motoneuron anatomy, whereas two thirds of this reduction (i.e., input resistance decrease by additional 20%) is due to a decrease in the specific membrane resistance of the motoneuron membrane. The specific membrane resistance represents the number of leak channels available in a patch of cell membrane and its decrease means that there are more ion channels inserted into the membrane (i.e., the cell has higher conductance). Comparison of WT and mutant motoneuron models

afterhyperpolarization, ↑↑: increase, ↓↓: decrease, --: no change.

motoneuron pathophysiology in these ALS transgenic models.

Higher & briefer spikes Faster rise & decay rates

Smaller & broader spikes Slower rise & decay rates

**size Rin AP/AHP PIC Gain Excitability** 

↑↑ ↑↑/-- Hyperexcitability


hypoexcitability

2004; Kuo et al., 2005; Zona et al., 2006; Pieri et al., 2009), but not in the slice (Quinlan et al., 2011) (Table 1). In the G85R transgenic model, the gain of mutant motoneurons was consistently reduced relative to WT (Bories et al., 2007; Pambo-Pambo et al., 2009). In the G93A (low expressor line) transgenic model, the gain of mutant motoneurons was increased in the slice preparation (Pambo-Pambo et al., 2009), but did not change in the whole-cord preparation (Filipchuk et al., 2010). The disagreement in reports on the change in motoneuron gain in the various transgenic models could be partially due to the variation in tissue preparation and extracellular Ca2+ concentration. Motoneuron dendrites have active conductances, which influence the motoneuron gain. Therefore, the portion of the motoneuron dendrites available during recording in the slice preparation would indirectly affect the measurement of the motoneuron gain in WT and mutant motoneurons. Also, the effect of extracellular Ca2+ concentration on the amplitude of Ca2+–activated and Ca2+–gated currents would influence the motoneuron gain measurement based on the degree of existence of these channels in WT and mutant motoneurons. Taken collectively, the change in gain and firing activity of mutant motoneurons is inconsistent within and across the various ALS transgenic models.

### **3.5 Motoneuron excitability in the various ALS transgenic models**

Despite the discrepancy in data on the excitability of mutant motoneurons in the various ALS transgenic models, some insights could be attained from the trend in electrical properties change (Table 3). In the G93A (high expressor line) model, most changes in electrical properties push toward increased excitability of mutant motoneurons. For instance, the increases in action potential height and rise and decay rates and the reduction in action potential width are signs of elevated transient and persistent Na+ currents that enhance the motoneuron excitability of mutant motoneurons. Also, the increase in persistent inward current amplitude further acts to enhance the motoneuron excitability. Collectively, these mechanisms push toward increased excitability of mutant motoneurons (i.e., excitability enhancement mechanisms). On the other hand, the increase in motoneuron size and reduction in input resistance are mechanisms that counteract increased excitability (i.e., excitability suppressive mechanisms) because the motoneuron becomes harder to recruit. It is unclear whether the excitability enhancement or suppressive mechanisms are disease or compensatory processes; however, the net effect of these mechanisms is increased excitability (i.e., hyperexcitability) of mutant motoneurons in the G93A (high expressor line) model (Table 3). This supposition is supported by the numerous reports on increased gain, although from cultured motoneurons, of mutant motoneurons in this transgenic model.

In the G93A (low expressor line) model, all changes in electrical properties indicate a reduction in the excitability of mutant motoneurons (i.e., hypoexcitability) (Table 3). These changes involve increase in motoneuron size, reduction in input resistance, smaller and broader action potential spikes, and slower rise rate (Table 3). The last two observations are indicative of a decrease in transient and persistent Na+ currents in mutant motoneurons relative to WT. In contrast to these data, the gain of mutant motoneurons measured using long pulses was higher than that of WT motoneurons; however, the gain measured using current ramp was not different from WT motoneurons (Pambo-Pambo et al., 2009). Taken collectively, more evidence is available on reduced excitability of mutant motoneurons in the G93A (low expressor line).

2004; Kuo et al., 2005; Zona et al., 2006; Pieri et al., 2009), but not in the slice (Quinlan et al., 2011) (Table 1). In the G85R transgenic model, the gain of mutant motoneurons was consistently reduced relative to WT (Bories et al., 2007; Pambo-Pambo et al., 2009). In the G93A (low expressor line) transgenic model, the gain of mutant motoneurons was increased in the slice preparation (Pambo-Pambo et al., 2009), but did not change in the whole-cord preparation (Filipchuk et al., 2010). The disagreement in reports on the change in motoneuron gain in the various transgenic models could be partially due to the variation in tissue preparation and extracellular Ca2+ concentration. Motoneuron dendrites have active conductances, which influence the motoneuron gain. Therefore, the portion of the motoneuron dendrites available during recording in the slice preparation would indirectly affect the measurement of the motoneuron gain in WT and mutant motoneurons. Also, the effect of extracellular Ca2+ concentration on the amplitude of Ca2+–activated and Ca2+–gated currents would influence the motoneuron gain measurement based on the degree of existence of these channels in WT and mutant motoneurons. Taken collectively, the change in gain and firing activity of mutant motoneurons is inconsistent within and across the

Despite the discrepancy in data on the excitability of mutant motoneurons in the various ALS transgenic models, some insights could be attained from the trend in electrical properties change (Table 3). In the G93A (high expressor line) model, most changes in electrical properties push toward increased excitability of mutant motoneurons. For instance, the increases in action potential height and rise and decay rates and the reduction in action potential width are signs of elevated transient and persistent Na+ currents that enhance the motoneuron excitability of mutant motoneurons. Also, the increase in persistent inward current amplitude further acts to enhance the motoneuron excitability. Collectively, these mechanisms push toward increased excitability of mutant motoneurons (i.e., excitability enhancement mechanisms). On the other hand, the increase in motoneuron size and reduction in input resistance are mechanisms that counteract increased excitability (i.e., excitability suppressive mechanisms) because the motoneuron becomes harder to recruit. It is unclear whether the excitability enhancement or suppressive mechanisms are disease or compensatory processes; however, the net effect of these mechanisms is increased excitability (i.e., hyperexcitability) of mutant motoneurons in the G93A (high expressor line) model (Table 3). This supposition is supported by the numerous reports on increased gain, although from cultured

In the G93A (low expressor line) model, all changes in electrical properties indicate a reduction in the excitability of mutant motoneurons (i.e., hypoexcitability) (Table 3). These changes involve increase in motoneuron size, reduction in input resistance, smaller and broader action potential spikes, and slower rise rate (Table 3). The last two observations are indicative of a decrease in transient and persistent Na+ currents in mutant motoneurons relative to WT. In contrast to these data, the gain of mutant motoneurons measured using long pulses was higher than that of WT motoneurons; however, the gain measured using current ramp was not different from WT motoneurons (Pambo-Pambo et al., 2009). Taken collectively, more evidence is available on reduced excitability of mutant motoneurons in

various ALS transgenic models.

the G93A (low expressor line).

**3.5 Motoneuron excitability in the various ALS transgenic models** 

motoneurons, of mutant motoneurons in this transgenic model.


Table 3. Summary of changes in motoneuron properties in the various ALS transgenic models. MN: motoneuron, Rin: input resistance, AP/AHP: action potential and afterhyperpolarization, ↑↑: increase, ↓↓: decrease, --: no change.

In the G85R transgenic model, not many changes were reported in the electrical properties of mutant motoneurons; however, these changes were consistent with reduced excitability and are supported by the decrease in gain of mutant motoneurons (Table 3). In conclusion, motoneuron pathophysiology is different in the various transgenic mouse models of ALS and their excitability varies from hypo- to hyperexcitability. This disparity could be related to the number of copies of the human SOD1 gene or the severity of the disease (transgenic models with high copy number of SOD1 genes have more aggressive ALS than transgenic models with low copy number of SOD1 genes). Transgenic models with a low number of SOD1 copies (e.g., G85R and G93A-low expressor line) showed a tendency for reduced excitability of mutant motoneurons, whereas transgenic models with a high number of SOD1 copies (e.g., G93A-high expressor line) showed a tendency for increased excitability of mutant motoneurons. These differences should be considered when studying the motoneuron pathophysiology in these ALS transgenic models.
