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. 2017 Mar 29;118(1):93–102. doi: 10.1152/jn.00045.2017

PICs in motoneurons do not scale with the size of the animal: a possible mechanism for faster speed of muscle contraction in smaller species

Seoan Huh 1, Ramamurthy Siripuram 4, Robert H Lee 4, Vladimir V Turkin 5, Derek O’Neill 5, Thomas M Hamm 5, Charles J Heckman 1,2,3, Marin Manuel 1,6,✉
PMCID: PMC5494365  PMID: 28356469

The small size of the mouse warrants concern over whether the properties of their neurons are a scaled version of those in larger animals or instead have unique features. Comparison of spinal motoneurons in mice to cats showed unique features. Firing rates in the mouse were much higher, in large part due to relatively larger persistent inward currents. These differences likely reflect adaptations for controlling much faster muscle fibers in mouse than cat.

Keywords: persistent inward currents, voltage clamp, electrical properties, adult spinal motoneurons

Abstract

The majority of studies on the electrical properties of neurons are carried out in rodents, and in particular in mice. However, the minute size of this animal compared with humans potentially limits the relevance of the resulting insights. To be able to extrapolate results obtained in a small animal such as a rodent, one needs to have proper knowledge of the rules governing how electrical properties of neurons scale with the size of the animal. Generally speaking, electrical resistances of neurons increase as cell size decreases, and thus maintenance of equal depolarization across cells of different sizes requires the underlying currents to decrease in proportion to the size decrease. Thus it would generally be expected that voltage-sensitive currents are smaller in smaller animals. In this study, we used in vivo preparations to record electrical properties of spinal motoneurons in deeply anesthetized adult mice and cats. We found that PICs do not scale with size, but instead are constant in their amplitudes across these species. This constancy, coupled with the threefold differences in electrical resistances, means that PICs contribute a threefold larger depolarization in the mouse than in the cat. As a consequence, motoneuronal firing rate sharply increases as animal size decreases. These differences in firing rates are likely essential in allowing different species to control muscles with widely different contraction speeds (smaller animals have faster muscle fibers). Thus from our results we have identified a possible new mechanism for how electrical properties are tuned to match mechanical properties within the motor output system.

NEW & NOTEWORTHY The small size of the mouse warrants concern over whether the properties of their neurons are a scaled version of those in larger animals or instead have unique features. Comparison of spinal motoneurons in mice to cats showed unique features. Firing rates in the mouse were much higher, in large part due to relatively larger persistent inward currents. These differences likely reflect adaptations for controlling much faster muscle fibers in mouse than cat.

most studies of the properties of neurons are carried out in rodents, either rat or mouse. Yet these species are very small compared with humans; for example, an adult mouse weighs rarely more than 40 g (Center for Genome Dynamics 2009), whereas humans average more than 70 kg, a more than 1,000-fold difference. Thus it would be expected that neuron size scales with animal size. The issue of size is particularly important for the motoneurons in the brain stem and spinal cord that provide all motor output. Comparison of lumbar motoneuron diameters shows a clear monotonic decrease in size as animals get smaller (Ishihara et al. 2001): cat motoneurons have an average diameter of 58 μm (Ulfhake and Cullheim 1981), rat motoneurons average 35 μm (Chen and Wolpaw 1994), and mouse motoneurons average 26 μm (McHanwell and Biscoe 1981). This decrease in size is paralleled with a decrease in contraction time of the muscle fibers that are innervated by these motoneurons (Meehan et al. 2010). It is also striking that, within a single species, there exists a wide range of motoneuron sizes, even for the pool of motoneurons innervating an individual muscle (Burke 1981). This range within a pool is the basis of Henneman’s size principle of recruitment, in which the smallest motoneurons are activated first (Henneman et al. 1965). The mechanism of this size based recruitment sequence is that smaller motoneurons have lower input conductances and hence require smaller synaptic currents to generate sufficient depolarization to attain the voltage threshold for spike initiation. Differences in size across species are presumably superimposed on this within-species range. These average size differences are matched by differences for input conductances, which are about one-third lower in the mouse compared with the cat (e.g., compare Lee and Heckman 1998b and Manuel et al. 2009; also see results).

The relation between size, conductance, and current may also apply across species. Hence, the smaller the animal, the smaller the current intensity required to attain a given level of depolarization. Thus, for motoneuron electrical behavior to be similar across species, currents should be much smaller in the mouse than in the cat. In this study, we systematically examined electrical properties of spinal motoneurons in anesthetized cat and mouse using in vivo voltage-clamp methods. We uncovered a striking exception to the expectation of smaller currents in smaller animals from our measurements of the persistent inward currents (PICs) that are essential for sustained motor output.

PICs are activated near spiking threshold and inactivate very slowly. As a consequence, they play a pivotal role in shaping the input-output relationship of motoneurons. PICs in spinal motoneurons, which are a mixture of L-type calcium current (CaPIC) and persistent sodium current (NaPIC) (Hounsgaard and Kiehn 1989; Lee and Heckman 2001; Li and Bennett 2003), have initially been described as currents able to produce sustained depolarizations, i.e., plateau potentials, in response to a brief depolarizing stimulus (Bennett et al. 1998a; Lee and Heckman 1998b). PICs are therefore ideally suited to generate sustained motor output required by motor units in antigravity muscles. Moreover, their dendritic localization also confers PICs with the ability to both amplify and prolong the responses of motoneurons to synaptic inputs (Bennett et al. 1998b; Manuel et al. 2007; Powers et al. 2012). Without PICs, distal synaptic inputs would have little to no effect on motoneuron output (Heckman and Binder 1991). In addition, the persistent component of the sodium current is essential in generating a sustained discharge of a motoneuron (Harvey et al. 2006; Kuo et al. 2006).

Most direct voltage-clamp recordings of PICs have been conducted in cat (Lee and Heckman 1998a) and turtle hindlimb motoneurons (Svirskis and Hounsgaard 1997) as well as rat hypoglossal (Powers and Binder 2003), sacral, and hindlimb motoneurons (Hamm et al. 2010; Li and Bennett 2003). Direct measurements of PICs in mouse hindlimb motoneurons heretofore have not been available due to the technical difficulties inherent with this small animal. In this study we have overcome these difficulties and adapted the in vivo voltage-clamp methods used in the cat and rat to the mouse. This study systematically compares the properties of PICs and other essential electrical behaviors in the mouse and cat. Our findings show that even though input conductance is progressively smaller from cat to mouse, PIC amplitudes remain constant across species. As a result, the mouse motoneurons tend to fire at significantly higher rates compared with cat motoneurons, likely to match the faster muscle contraction speed found in smaller sized mammals.

MATERIALS AND METHODS

In Vivo Experiments

Mouse.

In vivo experiments were conducted on B6SJL mice (postnatal days 30–140) of either sex. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Northwestern University. The surgical procedures have been described previously (Iglesias et al. 2011; Manuel and Heckman 2011; Manuel et al. 2009; Manuel et al. 2012) Briefly, atropine (0.2 mg/kg) mixed in perfusion solution (4% glucose, 1% NaHCO3, and 14% Hetastarch, pH 7) was given subcutaneously at the onset of the experiment to prevent salivation. After 10 min, anesthesia was administered intraperitoneally with pentobarbital sodium (70 mg/kg; Nembutal). After the absence of noxious reflex was confirmed, tracheotomy was performed and the mouse was artificially ventilated with 100% oxygen using the SAR-830/AP ventilator (CWE, Ardmore, PA). The end-tidal Pco2 was continuously monitored and maintained at around 4% (MicroCapstar; CWE). A rectal temperature probe was placed and temperature was kept around 37°C. The heart rate was monitored (CT-1000; CWE) and maintained between 400 and 500 beats/min. A catheter was placed in the jugular vein for supplemental anesthesia (6 mg/kg, every 10–15 min) mixed in perfusion solution. Depth of anesthesia was assessed by a lack of noxious reflexes, a stable heart rate, and a stable end-tidal Pco2, with no signs of resistance shown against the artificial ventilation. The biceps femoris muscle was then dissected out to reveal the sciatic nerve, which was then placed on a bipolar stimulating electrode. The muscle tissue and the nerve were covered with mineral oil to prevent dehydration. The vertebral column was immobilized with two pairs of horizontal bars (Cunningham Spinal Adaptor; Stoelting, Dublin, Ireland) applied on the T12 and L2 vertebral bodies, and the L2–L4 spinal segments were exposed by a laminectomy at the T13–L1 level. A custom-made chamber was fit around the exposed spinal segments and silicon sealant (WPI, Sarasota, FL) was applied to create a recording chamber. This chamber was filled with mineral oil to prevent dehydration of the spinal cord. Dura mater was carefully removed from the exposed spinal segments using no. 5 forceps. The motoneurons were impaled with micropipettes filled with 3 M KCl (resistance R = 10–12 MΩ). Intracellular recordings were obtained with an Axoclamp 2B amplifier and Spike2 software (CED, Cambridge, UK). All recordings were obtained using the discontinuous current or voltage clamp (6–7 kHz) and sampled at 20 kHz.

Cat.

Details of cat in vivo procedures have been published previously by Lee and Heckman (1998a, 1998b). All experiment protocols were approved by the respective Georgia Tech, Emory University, and Northwestern University Institutional Animal Care and Use Committees. Briefly, anesthesia was initiated with pentobarbital sodium (65 mg/kg). A tracheotomy was performed for subsequent artificial ventilation. Cannulas were inserted to the carotid artery for monitoring of blood pressure and to the jugular vein for fluid and drug administration (6.5 mg/kg). The cat then was immobilized with a vertebral clamp at the L3 vertebral process and with pins inserted into the hip. Temperature was continuously monitored via thermistors inserted into the esophagus and in the hindlimb mineral oil pool and maintained at 36–39°C using heat lamps and a heated water blanket. The expired CO2 level was also monitored and maintained around 4% by adjusting ventilation rates. The nerves to medial gastrocnemius and lateral gastrocnemius–soleus muscles were identified and carefully exposed and placed on bipolar electrodes. The exposed leg muscles and nerves were covered with a pool of mineral oil. A laminectomy was then performed at the L4–S1 level, and the exposed spinal cord was covered with mineral oil. A portion of the L7 dorsal root was placed on a monopolar electrode to measure the dorsal root volley. Microelectrodes were made using glass capillary electrodes with tip resistance of 5–15 MΩ. Electrodes were filled with 2 M potassium citrate. All records were low-pass filtered (−3 dB point of 10 kHz) and digitized at 10 kHz.

Electrophysiology

Mouse.

Each motoneuron was identified by the presence of an antidromic action potential from stimulation of the sciatic nerve. Frequency-current (f-I) relationship was determined on the basis of firing produced by a triangular current injection (1–10 nA/s) under discontinuous current-clamp configuration with switching varying between 6 and 8 kHz. The instantaneous firing frequency was then plotted against the intensity of the injected current. Ion and Ioff are currents at which the first action potential fires on the ascending ramp and the last action potential on the descending ramp, respectively. The f-I curve displayed two distinct regions: a nonlinear and highly variable subprimary range (SPR), followed by a linear region dubbed the primary range (PR). The gain of the f-I relationship was determined as the slope of the regression line on the PR (see dashed lines on the f-I curves of Fig. 1). Vthreshold was determined as the voltage where the first derivative of the voltage trace reached 10 mV/ms (Sekerli et al. 2004). Cells with unstable resting membrane potential or with action potential amplitude of <50 mV were excluded from analysis.

Fig. 1.

Fig. 1.

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Examples of current-voltage (I-V) and frequency-current (f-I) relationships in adult motoneurons. A1 and B1: raw current traces from 2 mouse motoneurons, recorded in response to voltage ramps at 5 mV/s. A2 and B2: overlay of I-V relationships. Ascending ramp is represented by black lines, descending ramp by gray lines, and leak current by dashed lines. A3 and B3: leak-subtracted I-V relationship. PIC amplitude is measured between dashed lines. A4 and B4: responses to triangular current ramps. Horizontal black bars at top indicate the regions that are enlarged in A5 and B5. A5 and B5: closer look at subprimary range. Arrows point to subthreshold oscillations. A6 and B6: f-I relationship. Ascending ramp is represented by black lines and descending ramp by gray lines; dashed line corresponds to the slope of the primary range (shown on ascending ramp only). C and D show examples of I-V and f-I relationships in 2 adult cat motoneurons. C1 and D1: overlay of I-V relationships. Ascending ramp is represented by black lines, descending ramp by gray lines, and leak current by dashed lines. C2 and D2: leak-subtracted I-V relationship. Amplitude is measured between dashed lines. C3 and D3: responses to triangular current ramps, with ramp sizes varied between 10 and 30 nA. C4 and D4: f-I relationship. Ascending ramp is represented by black lines and descending ramp by gray lines; dashed line corresponds to the slope of the primary range (shown on ascending ramp only).

PICs were recorded in single-electrode discontinuous voltage clamp (SEDVC; 6–8 kHz). Voltage clamp was attempted only in cells with stable membrane potential and in which the electrode capacitance could be adequately compensated. Clamp feedback gain was between 0.3 and 1.5. In addition to the feedback gain from the Axoclamp 2B amplifier (Axon instruments), an additional low-frequency feedback loop with a gain of 11 and a cutoff of −3 dB at 0.3 kHz was used to improve voltage control (Lee and Heckman 1998a). Monitor outputs were observed at all times to ensure reasonable settling of the electrode. To record PICs, a slow triangular voltage ramp (rate 5 mV/s) was applied. The current was then plotted against injected voltage to generate the current-voltage (I-V) function. Leak current was determined by fitting a regression line through the linear, subthreshold region of the I-V function, and the slope of this regression line was taken as input conductance (Gin). In some cases, it was difficult to identify an obvious linear region on the subthreshold I-V curve (see Fig. 1, B2 and B3). In these cases, the leak conductance was measured over the region between rest and 10 mV above rest, and extrapolated from there. Leak current was then subtracted from the total function, and this leak-subtracted I-V function was used to determine the PIC onset, offset, and amplitude (see Fig. 1, A2 and A3). PIC onset was defined as the current level at which the leak-subtracted I-V function first deviated from slope of zero. PIC offset was determined in a similar fashion, i.e., the current level just before the slope plateaued to zero on the descending ramp. The amplitude of PIC was calculated by measuring the total current from PIC onset or offset to the minima of the leak-subtracted I-V function.

Cat.

The hindlimb nerves were stimulated, and motoneurons were identified by the presence of an antidromic action potential. Discontinuous current-clamp mode (Axoclamp 2A amplifier; Axon instruments) was used to obtain f-I relations. Switching varied between 6 and 15 kHz. A triangular ramp of 10–30 nA for 10 s was injected into the cell, and the instantaneous firing frequency was plotted against current injected. For voltage clamp, SEDVC was used. The feedback gain ranged from 10 to 40 nA/mV, and an additional low-frequency feedback loop (gain of 100, −3 dB at 3 Hz) was used. Voltage-clamp data were filtered at 3 kHz, digitized at 5 kHz, and stored for further analysis. To measure PICs, a slow triangular voltage ramp was used with a rise time of 5 s, total duration of 10 s, and amplitude of 40 mV. Most cells were well controlled, producing only a few breakthrough spikes, and any cell with more than five breakthrough spikes was rejected from further analysis. Similarly to the calculations performed in mouse motoneurons, leak current was subtracted from I-V functions to measure the amplitude of PICs.

Data Analysis

Electrical recordings from 37 mouse motoneurons and 44 cat motoneurons that met all the inclusion criteria were analyzed for correlations and comparisons. Electrical properties for comparison tests were first confirmed for normal distribution using the D’Agostino-Pearson omnibus normality test. Unless otherwise specified, the unpaired t-test was used to compare averages for normally distributed data sets, and the nonparametric Mann-Whitney test was used to compare averages of non-normally distributed data sets. Pearson’s correlation coefficient was used for pairwise correlations. All statistics were calculated using Prism software (GraphPad Software, La Jolla, CA).

RESULTS

Successful voltage- and current-clamp recordings were obtained from 37 mouse hindlimb motoneurons and 44 cat motoneurons. Of 37 mouse motoneurons, 9 were not able to fire repetitively to the triangular current ramp. All 44 cat motoneurons had rhythmic firing, since any cat motoneuron that could not fire repetitively was abandoned before recording and thus precluded from this study.

Basic I–V and f-I Relationship

PICs recorded from adult mouse hindlimb motoneurons were similar in many respects to those recorded from anesthetized cats. Examples of mouse and cat PICs and their corresponding I-V and f-I relations are shown in Fig. 1, A–D. Cell A is a typical example of a mouse lumbar motoneuron with rhythmic firing. The ascending f-I relationship (Fig. 1A6) shows a steep increase in firing rate with high variability, considered as the subprimary range (SPR), and then a more linear increase in firing rate, noted as the primary range (PR). Consistent with observations by Delestrée et al. (2014), subthreshold oscillations (arrows in Fig. 1, A5 and B5) were prominent in the SPR, which contributed to the delay in the subsequent spike, leading to the high variability in frequency in SPR. Like most cells (23 of 30, or 76% of all rhythmically firing motoneurons recorded), this motoneuron had a recruitment current that is lower than its derecruitment current and therefore exhibited a clockwise hysteresis. Figure 1, A1–A3, shows the I-V relationship for the same motoneuron. Like most cells, it displayed an inward deviation from the leak conductance but did not have a negative slope region (NSR) and therefore had a relatively small PIC. Cell B, in comparison, had a larger PIC amplitude and a slightly higher f-I gain in the PR (defined by the slope of the gray dotted line in Fig. 1B6). Cells C and D are examples of typical cat motoneuron recordings. Cell C had a small PIC (Fig. 1C2) and a clockwise hysteresis (Fig. 1C4), similar to the mouse motoneuron shown in cell A. Cell D had a bigger PIC amplitude (Fig. 1D2) and a slightly larger f-I gain in the PR compared with cell C (Fig. 1D4).

PIC and Rhythmic Firing

The overall range of Gin (0.16–0.88 μS) in mouse motoneurons from this study is similar to values reported by Delestrée et al. (2014) (0.10–0.77 μS) with an average of 0.41 ± 0.15 μS. In contrast, Gin from cat motoneurons were 0.52–2.17 μS with an average of 1.22 ± 0.41 μS, almost three times larger than that of mouse motoneurons (P < 0.0001; Fig. 2A). Despite this large difference in Gin between cat and mouse motoneurons, PIC amplitudes on both ascending and descending ramps did not exhibit similar disparities; in fact, PIC amplitudes of cat and mouse motoneurons were not significantly different as can be seen in Fig. 2B (mouse: up 2.39 ± 1.96 nA, down 2.64 ± 2.57 nA, n = 30; cat: up 3.38 ± 4.47 nA, down 4.52 ± 4.75 nA, n = 44; P = 0.22, Kruskal-Wallis test). Further analysis revealed that cells with low input conductance had a tendency to have smaller PIC amplitudes, whereas those with higher input conductance on average had larger PIC amplitudes (mouse, Fig. 2, C and D; cat, Fig. 2, E and F).

Fig. 2.

Fig. 2.

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PIC amplitude scale with input conductance (Gin). A: comparison of input conductances between mouse and cat lumbar motoneurons (*P < 0.0001). The box upper and lower limits are the 75th and 25th quartiles, respectively. Each box is divided by the median. The whiskers extend all the way to the highest and lowest data points. B: comparison of PIC amplitudes in mouse (squares) and cat (circles) motoneurons. On both ascending and descending voltage ramps, PIC amplitudes were not significantly different between mouse and cat motoneurons. Box plot definitions are the same as in A. C: PIC amplitude (ascending ramp) vs. Gin (R2 = 0.20, P = 0.0131) in mouse motoneurons. D: PIC amplitude (descending ramp) vs. Gin (R2 = 0.15, P = 0.0339) in mouse motoneurons. E: PIC amplitude (ascending ramp) vs. Gin (R2 = 0.16, P = 0.0079) in cat motoneurons. F: PIC amplitude (descending ramp) vs. Gin (R2 = 0.10, P = 0.0334) in cat motoneurons. Both mouse and cat motoneurons with larger input conductance tend to have larger PIC values in ascending as well as descending I-V relationships.

Occasionally, there were motoneurons that could not fire repetitively but were still able to fire one or two action potentials at the onset of current steps (Fig. 3Av). The ability to fire a few action potentials to current steps indicated that the inability to repetitively fire was not due to deterioration of recording conditions or overall health of the cell. As mentioned earlier, cat motoneurons that could not repetitively fire were not recorded, and thus no comparison could be made. When mouse motoneurons that could repetitively fire were compared with ones that could not, motoneurons that did not exhibit rhythmic firing had a significantly larger average input conductance (0.61 ± 0.14 μS) compared with cells that had rhythmic firing (0.41 ± 0.15 μS; P = 0.02; Fig. 3B), similar to the findings of Delestrée et al. (2014). Cells that did not exhibit rhythmic firing had on average smaller PIC amplitudes (1.2 ± 0.5 nA for ascending ramp, 1.2 ± 0.7 nA for descending ramp, n = 9) compared with cells with rhythmic firing (2.4 ± 1.9 nA for ascending ramp, 2.6 ± 2.6 nA for descending ramp, n = 30), although this difference was statistically not significant (P = 0.17 for ascending ramp, P = 0.09 for descending ramp; Fig. 3C). This could be due to the fact that each motoneuron may need a different amount of current depending on its size (Gin), so we normalized each motoneuron’s PIC amplitude to its corresponding Gin. Comparison of normalized PIC amplitudes revealed that cells without rhythmic firing indeed had much smaller PIC per membrane area (approximated by Gin) compared with cells that could repetitively fire (P = 0.021 for ascending ramp, P = 0.036 for descending ramp; Fig. 3D). This result is consistent with the idea that PICs are essential for rhythmic firing of motoneurons (Kuo et al. 2006).

Fig. 3.

Fig. 3.

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Cells that do not fire repetitively have smaller PICs. A: example I-V relationship of a cell that is not able to repetitively fire on a current step protocol. i, Voltage ramps, with acceleration at 5 mV/s. ii, Overlay of I–V relationship. Ascending ramp is represented by black lines, descending ramp by gray lines, and leak current by dashed lines. iii, Leak-subtracted I-V relationship. PIC amplitude is measured under the dashed line. iv, Triangular current ramp elicited no repetitive firing. v, Current step protocol elicited only 2 action potentials. B: comparison of Gin of cells that are able to repetitively fire (squares) and of cells that are not able to repetitively fire (diamond). Gin of cells that are not able to fire repetitively is significantly larger than that of cells that are able to fire repetitively (*P = 0.022). C: comparison of PIC amplitude, on both ascending and descending ramp, of cells that could repetitively fire with ones that could not. Cells that are able to fire repetitively had larger amplitudes than cells that could not, but this slight increase was not statistically significant. D: comparison of PIC amplitude normalized to Gin between cells that are able to repetitively fire (squares) and cells that are not able to repetitively fire (diamonds). In both ascending and descending ramps, normalized PIC amplitude are larger in cells that are able to repetitively fire (*P = 0.021 and *P = 0.036, respectively). Box plot definitions in B–D are the same as in Fig. 2.

Relationship Between PIC Characteristics and Firing Behaviors

In cat motoneurons recorded in decerebrate cat preparations, analysis of I-V and f-I functions showed a significant correlation between current levels at PIC onset and offset and current levels at ascending and descending thresholds of f-I functions (Lee and Heckman 1998a, 1998b). As shown in Fig. 4A, I-PIC onset (dash-dotted line) is defined as the current level at which the I-V relation deviates from the linear passive leak conductance (dashed line). I-PIC max is the total current at the voltage where maximum PIC amplitude is measured (dotted line). Similarly, a straight line is fitted through the primary range (dashed line in Fig. 4B), and the frequency at which the f-I relationship deviates from this line is determined as the switch frequency from SPR to PR (dash-dotted line). Indeed, there was a strong correlation between PIC onset, offset, and firing thresholds on f-I relationships in anesthetized cat motoneurons as shown in Fig. 4, D and F. Mouse motoneurons also showed significant correlation between PIC onset current and firing threshold current (R2 = 0.24, P = 0.0083; Fig. 4C). However, the relationship between offset current and descending threshold of the f-I function in mouse motoneurons was not significant (R2 = 0.09, P = 0.12). Interestingly, there was a strong correlation between current at which PIC peaks and current at which the f-I relation switches from SPR to PR in the mouse (R2 = 0.75, P < 0.0001; Fig. 4G) and cat (R2 = 0.64, P < 0.0001; Fig. 4H), suggesting that PICs play a role in setting the transition between SPR and PR.

Fig. 4.

Fig. 4.

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Relationships between PICs and firing characteristics. A: I-V relationship overlay. Ascending ramp is represented by black lines, descending ramp by gray lines, and leak current by dashed lines. Current at PIC onset (I-PIC onset) is represented by a dash-dotted arrow and I-PIC max by a dotted arrow. B: f-I relationship of the same motoneuron recorded in A. Ascending ramp is represented by black lines and descending ramp by gray lines, dotted line indicates the limit between the subprimary (SPR) and primary range (PR), and dashed line represents the slope of the PR. The firing frequency at the transition between the SPR and PR is indicated with a dash-dotted arrow. C: recruitment current (Ion) vs. I-PIC onset (R2 = 0.24, P = 0.0083) in mouse motoneurons. Onset of recruitment on a current ramp had a significantly positive correlation with current at PIC onset. D: Ion vs. I-PIC onset (R2 = 0.58, P < 0.0001) in cat motoneurons. Onset of recruitment on a current ramp had a significantly positive correlation with current at PIC onset. E: derecruitment current (Ioff) vs. current at I-PIC offset (R2 = 0.09, P = 0.1184) in mouse motoneurons. Offset of recruitment on a current ramp did not have a significant correlation with current at PIC offset. F: Ioff vs. I-PIC offset (R2 = 0.46, P < 0.0001) in cat motoneurons. Offset of recruitment on a current ramp had a significant correlation with current at PIC offset. G: current where PR starts in a f-I relationship vs. I-PIC peak (R2 = 0.75, P < 0.0001). Current where SPR switches to PR showed a significantly positive correlation with current where PIC peaks. Note that some cells were not able to reach the primary range of firing, which is why there are fewer points in F than in C and E. H: current where PR starts in a f-I relationship vs. I-PIC peak (R2 = 0.64, P < 0.0001). Current where SPR switches to PR showed a significantly positive correlation with current where PIC peaks.

Four distinct types of discharge patterns as shown in Bennett et al. (2001) were also found in mouse motoneurons recorded in this study. Type I motoneurons display a linear and symmetrical frequency response to current ramp injection with Ion and Ioff occurring at very similar or equivalent current intensities: ΔI = Ioff – Ion ≈ 0 (Fig. 1B6). Type II motoneurons exhibit much lower frequency of firing on the descending ramp of the current injection, lower Ion than Ioff, thus creating ΔI > 0 and producing a clockwise hysteresis (Fig. 1A6). Type III motoneurons display a linear frequency response to current ramp injection, but Ioff is typically lower than Ion, i.e., ΔI < 0 (not shown). Finally, type IV motoneurons are opposite of type II motoneurons and show an increase in firing rate on the descending ramp, with Ioff that is lower than Ion (ΔI < 0), producing a counterclockwise hysteresis (not shown). In examining which PIC characteristics contribute to these patterns of firing hysteresis, it was found that Gin and normalized PIC amplitude on the descending ramp had a moderate but significant correlation with ΔI (R2 = 0.20, P = 0.015; Fig. 5A). That is, the smaller the normalized PIC amplitude in a given motoneuron on the descending ramp, the more the f-I characteristics exhibited a clockwise hysteresis. In cat motoneurons, there was a similar but nonsignificant trend between ΔI and PIC on the descending ramp (R2 = 0.06, P = 0.19; Fig. 5B).

Fig. 5.

Fig. 5.

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Relationship between PIC and hysteresis. A and B: PIC amplitude on the descending I-V ramp, normalized by input conductance (Gin) plotted against ΔI (Ioff − Ion). The larger the PIC amplitude on the descending ramp, the more hysteresis shown in the f-I relationship in mouse motoneurons (A). This correlation did not reach statistical significance in cat motoneurons (B).

DISCUSSION

In this study, for the first time, we have examined mouse and cat spinal motoneurons using an in vivo voltage-clamp technique in anesthetized preparations. Whereas cat motoneurons are known to have firing behaviors closer to those of human subjects, a deeper understanding of mouse motoneurons is becoming more essential because many genetic models of human diseases that affect motor systems are being generated in mice. This systematic comparison of mouse and cat motoneurons will be able to set the basis of how motoneurons control motor systems in healthy states and further help us understand how disease states such as amyotrophic lateral sclerosis alter these properties.

Mouse Motoneurons Have a Larger PIC Than Expected

We found that despite the much larger size (approximated by Gin) of cat motoneurons compared with mouse motoneurons, PIC amplitudes were surprisingly similar. This similarity means that PICs in the mouse are much larger relative to their input conductances. Figure 6C shows mouse motoneurons had significantly (P < 0.0001) higher values for PIC/Gin (5.81 ± 3.87 nA/μS) compared with cat motoneurons (2.65 ± 3.41 nA/μS). Our results suggest that this larger relative PIC contributes to the significantly higher gain (P < 0.0001; Fig. 6A) in mouse motoneurons (average = 10.62 ± 5.69 Hz/nA) compared with cat motoneurons (average = 1.68 ± 0.47 Hz/nA). However, other factors might also contribute to this considerable difference in primary range (PR) gain between mouse and cat motoneurons, such as their difference in afterhyperpolarization (AHP) of action potentials (AHP duration: 46 ± 11 ms in mouse, 73 ± 22 ms in cat; Manuel et al. 2009) and membrane time constant [τm: 2.5 ± 1.0 ms in mouse (Manuel et al. 2009), 7.0 ± 1.5 ms in cat (Zengel et al. 1985)]. Both shorter AHP duration and a faster time constant contribute to faster firing frequency in mouse motoneurons. This faster firing is a necessary adaptation in motoneurons to match the faster muscle contraction speed in smaller animals compared with larger animals.

Fig. 6.

Fig. 6.

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Comparisons between cat, rat, and mouse motoneurons. A: f-I gain in primary range (PR) is highest in mouse motoneurons (average = 10.62 ± 5.69 Hz/nA, n = 17), followed by that in rat (average = 4.7 ± 3.3 Hz/nA, n = 37; data provided by T. M. Hamm) and then cat motoneurons (average = 1.68 ± 0.47 Hz/nA, n = 24). All differences were statistically significant (Krusal-Wallis followed by Dunn’s post hoc test) as indicated by asterisks. B: f-I gain in PR plotted against Gin in mouse (squares), rat (diamonds), and cat (circles) motoneurons. C: PIC amplitude normalized by Gin is significantly larger (*P < 0.0001) in mouse (average = 5.82 ± 3.87 nA/μS, n = 30) compared with cat motoneurons (average = 2.64 ± 3.42 nA/μS, n = 44). Box plot definitions in A and C are the same as in Fig. 2.

As anticipated, rat motoneurons had an input conductance intermediate between cat and mouse motoneurons (average = 0.5 ± 0.1 μS, n = 37; Turkin et al. 2010). Because f-I gain for rat motoneurons (average = 4.7 ± 3.3 Hz/nA, n = 37) also fell between values for mouse and cat motoneurons (Fig. 6, B and C), one would expect that Gin-normalized PIC amplitudes from rat motoneurons should also fall between those from mouse and cat motoneurons. When PIC amplitudes of rat motoneurons capable of repetitive discharge were examined, amplitude of rat PICs ranged from 0.54 to 18.27 nA (average = 7.7 ± 5.7 nA, n = 15) (Hamm et al. 2010; unpublished data, Hamm TM). Consequently, PIC normalized by Gin had an average of 14.2 ± 12.9 nA/μS, a value exceeding that of both mouse and cat motoneurons with high variability. This could be a result of the use of different anesthetics; rat recordings were obtained under ketamine-xylazine anesthesia instead of pentobarbital used in mouse and cat preparations. Because ketamine acts through NMDA receptors in contrast to pentobarbital, which works through potentiating GABAA receptors (Wakasugi et al. 1999), there could be differences in synaptic activity contributing to the difference in PIC amplitudes. Moreover, PIC amplitudes obtained under ketamine-xylazine anesthesia may be more comparable to those obtained in decerebrate than pentobarbital-anesthetized preparations (Button et al. 2006). The larger size of the PICs in mouse vs. cat motoneurons is unlikely to be due to a difference in depth of anesthesia. In both cases, recordings are obtained on deeply anesthetized animals, as assessed by the lack of noxious reflexes and the stability of heart rate and end-tidal Pco2. A deep anesthesia is particularly important for mouse recordings because the mice were not paralyzed, and any resistance of the mouse against the ventilator would have precluded obtaining stable recordings. One cannot exclude the possibility that pentobarbital does not reduce PICs to the same extent in rodents as in cats, for example, by causing less depression of serotoninergic pathways. However, some PICs recorded in rats under ketamine-xylazine were large (>10 nA), larger than most PICs recorded in mice and cats under pentobarbital anesthesia, which suggests that rodent PICs are indeed partially blocked by pentobarbital. In addition, Button et al. (2006) have shown than PICs recorded in rats under ketamine-xylazine were significantly reduced by subsequent injection of pentobarbital.

Another possibility is that mouse PIC measurements are contaminated by the Ih current. Indeed, Ih is a current activated at hyperpolarized potential and therefore contributes to the leak conductance. Because PIC amplitude is measured after the leak current is subtracted, a large Ih conductance can artificially inflate the amplitude of PICs compared with the measurements obtained if leak conductance is measured at more depolarized potentials, when Ih is deactivated (Li et al. 2007). However, the values of “sag ratios,” a measure of the strength of the Ih current, are not different between cat (on average 1.4), rat (1.15; unpublished data, Turkin VV, O’Neill D, and Hamm TM) and mouse motoneurons (1.2; Manuel et al. 2009). Therefore, despite the contamination of our PIC measurements by Ih, this error affects similarly cat and mouse motoneurons, and our conclusions remain the same. Taken together, these results for cat, rat, and mouse motoneurons show that PICs do not decrease with size.

Our technical advance was to adapt the voltage-clamp methods for larger mammals such as cats, which weigh around 3,000 g, to the much smaller adult mice, which weigh around 30 g on average. Thus we were able to directly compare properties of mouse and cat motoneurons for the first time in anesthetized preparations. Previous voltage-clamp studies were mainly carried out in slice preparations, which limited the age of spinal cords to those that were not too enlarged to withstand hypoxia during slicing preparations. The use of deep anesthesia and the ability to record from lumbar motoneurons using similar setups afforded us the chance for direct comparison between the two species. We carefully monitored variations in resting membrane potentials but found that recordings were as stable in mice as in cats, despite the smaller size of motoneurons in mice. A potential disadvantage of this voltage-clamp approach in all three species is that sharp electrodes are required to pass through the heavily myelinated surface of spinal cords to reach motoneurons in the ventral horn. Unlike the patch-clamp technique, sharp electrodes impose a risk of leakage due to electrode-induced shunt, thus making input conductance appear higher than its actual value. The electrodes used to record mouse motoneurons need to be significantly smaller (13–15 MΩ) than those used in cat experiments (1–2 MΩ) because of the difference in soma size. Gustafsson and Pinter (1984) estimated that the error on input conductance measurements in cats should be no larger than 30%. We estimate that in mouse motoneurons this value should not be greater than it is in the cat motoneurons given the smaller size of the sharp electrodes. In addition, should there be an extensive leakage greater than 30%, one would expect to see unstable recordings or stability progressively declining, which was never the case for all recordings that were included in our analysis.

Motoneuron firing patterns must be able to match the properties of the motor unit to which they belong. Previous studies in mouse, rat, and cat motoneurons suggested that there was an adaptation in speed matching between motor unit contraction time and action potential properties as one goes from larger to smaller mammals. This adaptation, however, was not merely size dependent. In other words, as animal size decreased, specifically from cat to rat to mouse, afterhyperpolarization (AHP) systematically became smaller compared with contraction time. Specifically, cat studies from Zengel et al. (1985), Cope et al. (1986), and Kernell (2006) showed twitch duration was matched with AHP duration, whereas Manuel and Heckman (2011) showed twitch duration was on average 30% longer than AHP duration in mouse motor units. When we compare the contraction speed alone among species, in cats it ranges from 16 to 30 ms, almost three to four times as long as that in mice (4–12 ms; Close 1972). Thus we believe that threefold scaling of AHP alone from mouse to cat motor units would not account for the scaling of the muscle contraction speed. Indeed, the striking finding in our study that even though there is at least a 3-fold difference in input conductance between mouse and cat motoneurons, and a 1.25-fold difference between mouse and rat motoneurons, PIC amplitudes stay in a relatively similar range, giving a huge scaling factor in PIC normalized by Gin among different animals. This, we believe, is the additional factor that drives the large scaling of PR gain among motoneurons from these animals. Because we observed no scaling of PIC amplitudes but PIC/Gin, we predict that ionic currents stay the same but the Gin-normalized currents are what actually scale.

Because PICs are strongly influenced by neuromodulators such as serotonin and norepinephrine, there is a potential impact of neuromodulation, or lack there of, considering the lower neuromodulatory state due to the use of an anesthetic agent. However, we presume this low neuromodulatory state to be similar in pentobarbital-anesthetized mice and cats, although it is likely higher in ketamine-xylazine-anesthetized rats. Questions remain whether mouse and cat motoneurons have the same level of facilitation to these monoamines. Murray et al. (2010) showed bath application of cyproheptadine, a 5-HT2 and NAα1 receptor inverse agonist, eliminated PIC-mediated plateau potential as well as self-sustained firing in sacral rat motoneurons, and we suspect that lumbar motoneurons would be similarly affected. It is clear that, to generate an equal amount of self-sustained firing in motoneurons of different species/sizes, PICs must scale up with size. Because mouse motoneurons have much lower conductances, smaller PICs are sufficient to induce the negative slope regions in their I–V functions that are necessary for generating a significant difference in their recruitment and derecruitment rates (Δf). However, it appears that the larger PICs needed in the cat are not achieved by having larger PICs in their basal state, but instead by having a larger effect of neuromodulation. In the cat, strong negative slopes only occurred in what the authors called the “enhanced state” (decerebrate plus methoxamine; Lee and Heckman 2000). Our present results show that PICs recorded in pentobarbital-anesthetized cats had an amplitude of 3.4 ± 4.5 nA, whereas in the enhanced state, they were 17.0 ± 2.7 nA (Lee and Heckman 2000). In contrast, there is strong self-sustained firing behavior in rat sacral motoneurons after chronic spinalization, where according to Harvey et al. (2006), PICs were 2.79 ± 0.94 nA compared with the acute spinal state amplitude of 1.1 ± 1.2 nA. Therefore it seems that although neuromodulation increases PIC amplitude by more than fivefold in cat motoneurons, it generates less than a threefold increase in rat sacral motoneurons, which suggests that neuromodulation has a greater effect on larger species (cat, human) vs. rodents.

Because our ultimate goal is to understand the basis of firing behavior of motoneurons across species and in disease states, it is important to note the differences that have already been reported in awake behaving animals. Ritter et al. (2014) found a wide range of firing frequencies ranging from 9 to 68 Hz in lateral gastrocnemius muscle of mice during quiet standing. In contrast, in rat motor units, this range was found to be 16–25 Hz in the soleus muscle (Eken 1998). Higher rates were reported in locomoting rats (Gorassini et al. 1999, 2000): 60–100 Hz in tibialis anterior and lateral and medial gastrocnemius, and 30 Hz in soleus. In cats, Hoffer et al. (1987) reported 15–25 Hz in serratus anterior, 40 Hz in rectus femoris, and 25–35 Hz in vastus medialis. Finally most soleus motor units recorded in humans during quiet standing are firing at a much lower rate (between 7 and 11 Hz; Eken 1998). If the scaling rules seen across mouse, rat, and cat hold for even larger species, then PIC amplitudes in humans may be similar to those in these animals, their input conductances should be much greater, and their PIC/Gin correspondingly less.

In summary, our results show that the lack of scaling of PICs between mouse and cat motoneurons is likely to be a primary mechanism of the tendency of the smaller animals to firing action potentials at a faster rate for a given amount of input. These differences are important in considering how to extrapolate the results from neurons in the mouse to an understanding of human motor output.

GRANTS

This work was supported by National Institutes of Health Grants R01NS089313, R01NS077863, and R01NS082463 and the National Science Foundation Graduate Research Fellowship Program (fellow ID 2012137838).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.H., C.J.H., and M.M. conceived and designed research; S.H., R.S., R.H.L., V.V.T., D.O., and T.M.H. performed experiments; S.H., R.H.L., V.V.T., D.O., and T.M.H. analyzed data; S.H., C.J.H., and M.M. interpreted results of experiments; S.H. and M.M. prepared figures; S.H. drafted manuscript; S.H., C.J.H., and M.M. edited and revised manuscript; S.H., R.S., R.H.L., V.V.T., D.O., T.M.H., C.J.H., and M.M. approved final version of manuscript.

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