Neurons in the brain communicate using chemical signals that they send and receive across junctions called synapses. To maintain normal behavior over time, circuits of neurons must reliably process these signals. A variety of nervous system disorders may result if they are unable to do so, as may occur when neural activity changes as a result of disease or injury.

The processes underlying the stability of a neuron’s synapses is referred to as “homeostatic” synaptic plasticity because the changes made by the neuron directly oppose the altered level of activity. In one form of homeostatic plasticity, known as synaptic scaling, neurons modify the strength of all of their synapses in response to changes in neural activity.

There is substantial experimental evidence to show that in young animals, neurons that communicate using a chemical called glutamate undergo synaptic scaling in response to artificial changes in activity. It had not been directly shown that synaptic scaling protects the neural activity of adult animals in their natural environments, in part, because neural activity in most healthy animals generally only goes through small changes. However, the neurons in the brain that cause the breathing muscles of bullfrogs to contract are ideal for studying homeostatic plasticity because they are naturally inactive for several months when frogs hibernate in ponds during the winter. During this time, the bullfrogs do not need to use their lungs to breathe because enough oxygen passes through their skin to keep them alive.

Santin et al. have now observed synaptic scaling of glutamate synapses in individual bullfrog neurons that had been inactive for two months. Further experiments that examined the activity of the breathing control circuit in the brainstem provided evidence that synaptic scaling leads to sufficient amounts of neural activity that would activate the breathing muscles when frogs emerge from hibernation. Therefore neural activity after prolonged, natural inactivity relies on synaptic scaling to preserve life-sustaining behavior in frogs.

These results open up new questions: mainly, how do synaptic scaling and other forms of homeostatic plasticity operate in animals as they experience normal variations in neural activity? Determining how homeostatic plasticity works normally in an animal will help us to understand what happens when plasticity mechanisms go wrong, as is thought to occur in several human nervous system diseases including nervous system injury, Alzheimer’s disease, and epilepsy.

For the brain to work properly, neurons must employ compensatory mechanisms to retain information in synapses and to maintain normal circuit function. These compensatory mechanisms are considered homeostatic if they regulate firing frequency and/or circuit activity around a set-point, but they also may oppose disturbances to neuronal function without being firmly homeostatic (Turrigiano, 2012). Homeostatic and compensatory plasticity maintains neuronal function through a suite of synaptic and intrinsic mechanisms (Pozo and Goda, 2010; Schulz and Lane, 2017; Turrigiano, 2011), with the ultimate goal being preservation of normal network function. The most prominent mechanisms used to counter disturbances in neuronal activity include synaptic scaling, a slow and global form of synaptic plasticity where excitatory synaptic strength increases or decreases equally across all synapses (Turrigiano et al., 1998), modulation of presynaptic neurotransmitter release (Davis, 2013), and altering voltage-gated membrane conductances (e.g. Na⁺, K⁺, and Ca²⁺ channels) (Desai et al., 1999; Ransdell et al., 2012; Wilhelm et al., 2009).

Manipulations that perturb neuronal activity in vitro are, by far, the most common experimental approaches to evoke homeostatic plasticity (Williams et al., 2013), leaving many open questions as to what normal scenarios require homeostatic mechanisms to regulate circuit output and behavior. Only few physiological examples exist. In developing rodents, neurons in the visual cortex undergo synaptic scaling at the onset of visual experience (Desai et al., 2002). Also within the visual system, neurons in the retinotectal circuit of the aquatic frog, Xenopus laveis, homeostatically decrease their intrinsic excitability to compensate for developmental increases in excitatory synaptic inputs (Pratt and Aizenman, 2007). Additionally, plasticity in the dynamic regulation of synaptic strength seems to compensate for variability in neuron number in the crustacean stomatogastric ganglion (Daur et al., 2012). However, rather than uncovering responses to normal, expected physiological challenges, most investigations of homeostatic plasticity dramatically perturb neuronal activity with artificial (e.g. pharmacological and genetic manipulations) or pathological (e.g. sensory denervation/stimulation, injury) modalities. While these synthetic challenges evoke striking compensatory or homeostatic responses in neurons in vivo (Aizenman et al., 2003; Braegelmann et al., 2017; Echegoyen et al., 2007; Frank, 2014; Gonzalez-Islas and Wenner, 2006; Hengen et al., 2013; Kline et al., 2007; Knogler et al., 2010; Lambo and Turrigiano, 2013; Rajman et al., 2017), how stabilizing mechanisms activated by these kinds of disturbances relate to non-pathological physiological scenarios is unclear, especially in mature animals. Animals with life histories involving profound disturbances to neuronal activity may provide new insights into the physiological necessity of such mechanisms underlying stability of neurons in functional circuits.

Here, we take advantage of an adult animal (American bullfrogs, Lithobates catesbeianus) that normally undergoes drastic and prolonged reductions in neuronal activity to understand if compensatory mechanisms commonly evoked during artificial deprivation of neuronal firing occur in an environmentally relevant setting. We demonstrate that a well-described mechanism of homeostatic plasticity, up-scaling of excitatory synapses, occurs in motoneurons innervating a primary respiratory muscle from bullfrogs after 2 months in a submerged-aquatic, overwintering habitat: a natural environment that induces complete respiratory motor inactivity (Santin and Hartzler, 2017). Strikingly, we further identify that increased excitatory synaptic strength onto these motoneurons enhances population motoneuron output from the respiratory network, thereby acting to preserve respiratory motor drive to critical respiratory muscles under conditions when breathing would be obligatory at warm temperatures after 2 months without breathing movements (Santin and Hartzler, 2016a). As failure to stabilize motor output from the brainstem to respiratory muscles has fatal consequences, these results implicate up-scaling of excitatory synapses on respiratory motoneurons as a critical mechanism for an adult vertebrate experiencing prolonged bouts of neural inactivity in a natural context.

American bullfrogs often overwinter in ice-covered ponds with no need to use their lungs for breathing during cold winter months due to adequate skin gas exchange at low temperatures (Tattersall and Ultsch, 2008). Although frogs retain a relatively high locomotor capacity during overwintering submergence (Tattersall and Boutilier, 1999), important muscles of the respiratory apparatus, buccal floor constrictors and glottal dilators, are inactive during cold-submergence (Santin and Hartzler, 2017). Despite months of respiratory motor inactivity, upon forced emergence at warm temperature bullfrogs immediately exhibit breathing movements, ventilate to match resting metabolic demands, increase ventilation during exposure to low oxygen, and generate respiratory motor output from the brainstem (Santin and Hartzler, 2016a; Santin and Hartzler, 2016b). Largely normal ventilatory behaviors after months of motor inactivity led us to hypothesize that preservation of respiratory motoneuron output from the brainstem to breathing muscles may rely on slow acting, global mechanisms that compensate for neuronal inactivity (i.e. homeostatic plasticity). To test this hypothesis, we backfilled vagal motoneurons that innervate a primary respiratory muscle, the glottal dilator involved in regulating airflow into and out of the lung in frogs (Gans et al., 1969), with fluorescent dye (Figure 1). We then measured synaptic and intrinsic neuronal properties from fluorescently labeled respiratory motoneurons in brainstem slices at 23°C (a temperature requiring lung breathing) in control bullfrogs and those that experienced ~2 months of respiratory motor inactivity during overwintering-like conditions. This allowed us to determine the extent to which winter inactivity leads to expression of compensatory mechanisms in respiratory motoneurons.

Figure 1

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Increases in excitatory synaptic transmission occur during inactivity/AMPA-glutamate receptor blockade in vitro (Fong et al., 2015; Turrigiano et al., 1998) and in vivo (Hengen et al., 2013; Knogler et al., 2010). Therefore, we first sought to determine whether winter inactivity results in a compensatory increase in excitatory synaptic transmission by assessing the quantal amplitude (i.e. the postsynaptic response to spontaneous release of neurotransmitter single vesicles recorded in voltage clamp, termed miniature excitatory post synaptic currents [mEPSCs]). Consistent with this hypothesis, the amplitude and charge transfer of mEPSCs mediated by AMPA-glutamate receptors were increased in respiratory motoneurons after overwintering inactivity (Figure 2A–D). In contrast, we did not observe changes in mEPSC frequency (Figure 2E), suggesting a similar number of glutamate release sites onto respiratory motoneurons in brainstem slices. Although the mEPSC amplitude is prone to dendritic filtering, charge transfer (i.e. mEPSC area) is more robust against space clamp errors (Spruston et al., 1993). Thus, increases in both amplitude and charge transfer of the mEPSC suggest an enhancement of post-synaptic AMPA receptor function rather than changes in dendritic filtering properties of motoneurons (Han and Stevens, 2009). Consistent with this assertion, parameters influencing and influenced by dendritic filtering, neuronal input resistance and mEPSC rise time, respectively, did not differ between control and winter inactivity motoneurons (Figure 2F–G). These results indicate that respiratory motoneurons have enhanced excitatory synaptic strength after winter inactivity presumably by up-regulating postsynaptic function of AMPA-glutamate receptors.

Figure 2

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Increased excitatory synaptic strength of neurons responding to reductions in activity typically follows a multiplicative scaling relationship in dissociated culture (Fong et al., 2015; Turrigiano et al., 1998) and in vivo (Knogler et al., 2010; Lambo and Turrigiano, 2013), implying that all measurable synapses increase in strength relatively by the same extent. Thus, synaptic scaling maintains relative synaptic strength in the face of disturbances in neuronal activity. This is observed experimentally as a right-shift in the cumulative distribution of mEPSC amplitudes induced by inactivity that can be down-scaled mathematically by dividing the entire distribution of mEPSCs by a common scaling factor. The scaling factor is derived from the slope of a linear rank order plot of mEPSC amplitudes from control and activity-deprived distributions. In contrast to synaptic scaling, a rank order plot following long-term potentiation (LTP) - a rapid and synapse-specific form of synaptic plasticity- is better approximated by an exponential curve (Gainey et al., 2009). Do increases in the mEPSC amplitude scale multiplicatively following winter inactivity, an ecologically relevant, long-term perturbation of neuronal activity?

When rank ordering an equal number of mEPSC amplitudes from control and winter inactivity vagal respiratory motoneurons (black dots; 50 mEPSCs from 16 neurons in each group), the plot is well-fit by a linear regression (r² = 0.99; Figure 3A; dashed blue line), rather than an exponential curve (r² = 0.73; Figure 3A; dashed black line). If the slope of this line is greater than 1 (i.e., the unity line; Figure 3A; solid gray line), it suggests multiplicative scaling has occurred. We detected a slope of 1.51. The slope of the line from the regression is equal to the scaling factor, implying that all measurable excitatory synapses increased by a factor of 1.51 during winter inactivity. Indeed, when the right-shifted cumulative distribution of mEPSC amplitudes from winter inactivity motoneurons (Figure 3B; solid blue line; Kolmogorov-Smirnov test control vs. winter inactivity; p=2.2×10⁻¹⁶) is divided by the scaling factor, the ‘down-scaled’ winter inactivity distribution (Figure 3B; dashed blue line) overlaps the control mEPSC amplitude distribution (Figure 3B; solid green line; Kolmogorov-Smirnov test control vs. scale winter inactivity; p=0.1403). Therefore, excitatory synapses on inactive respiratory motoneurons ‘scale up’ during winter inactivity.

Figure 3

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In addition to synaptic scaling, reduced activity may influence the function of intrinsic ion channels, including Na⁺, K⁺, Ca²⁺, and non-selective cation channels, to oppose reductions in neuronal and network activity. Compensatory changes in the intrinsic membrane currents carried by ion channels during inactivity typically manifest as enhanced excitability in response to injected current (Desai et al., 1999; Echegoyen et al., 2007; Lambo and Turrigiano, 2013; Wilhelm et al., 2009). Therefore, we evaluated action potential firing in respiratory motoneurons to determine whether intrinsic changes, in addition to synaptic scaling, may be involved in the compensatory response to winter inactivity. To assess intrinsic excitability, we measured firing frequency of respiratory motoneurons in response to step increases in injected current. Figure 4A shows example recordings of responses to current injections from control (green traces; top) and winter inactivity (blue traces; bottom) motoneurons. We did not observe differences in firing frequency at any amount of current (Figure 4B). The frequency-current (F-I) gain did not differ between both groups of motoneurons (Figure 4B–C). Additionally, resting membrane potential did not differ between control and winter inactivity motoneurons (control: −57.62 ± 1.74 mV vs. winter inactivity: −57.13 ± 1.41 mV; p=0.8269; T₃₀ = 0.2206). These results suggest that these motoneurons do not enhance their excitability in response to winter inactivity.

Figure 4

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Thus far, our results indicate that up-scaling of AMPA glutamate-receptors increases the synaptic strength of respiratory motoneurons in response to winter inactivity. How may this affect respiratory behavior immediately following overwintering? In bullfrogs and other vertebrate animals, central pattern generating (CPG) networks in the brainstem underlie the neural activity of respiratory movements (Baghdadwala et al., 2015; Feldman et al., 2013). This respiratory CPG activity is transmitted to cranial and spinal motoneurons, in part, via excitatory glutamatergic synapses (Greer et al., 1991; Kottick et al., 2013) to produce the motor output that activates respiratory muscles. We hypothesized that synaptic scaling on respiratory vagal motoneurons receiving respiratory CPG input could increase the reliance of AMPA-receptor transmission of the vagal respiratory motor outflow in bullfrogs after winter inactivity. To test this possibility, we extracellularly recorded respiratory-related vagal motoneuron population discharge from the cut nerve root (i.e. fictive breaths exiting the brainstem via the axons of vagal motoneurons) in rhythmically-active, in vitro brainstem-spinal cord preparations (Figure 5A); a preparation that produces respiratory motor output similar to that observed in intact bullfrogs (Hedrick, 2005). In rhythmic respiratory preparations, synaptic properties of motoneurons (and premotoneurons) can be determined by assessing relative changes in the amplitude of the integrated fictive breath after application of receptor antagonists, while the activity of the rhythm generating circuits can be assessed through the frequency of fictive breaths (for example Greer et al., 1991; Johnson et al., 2002). Using the same in vitro preparation from bullfrogs, Chen and Hedrick (2008) showed that a low concentration of AMPA-receptor antagonist does not influence the amplitude of fictive breaths, while the frequency is more sensitive to inhibition of AMPA-receptors. To test the hypothesis that winter inactivity enhances the reliance of AMPA-receptor transmission on vagal motoneurons and to explore the possibility of altered AMPA-receptors in rhythm generating circuits, we measured the amplitude and frequency of fictive-breaths via vagal motoneurons to assess differences in the sensitivity to an AMPA receptor antagonist, DNQX, between control and winter inactivity bullfrogs. To be consistent with our results demonstrating up-scaling of AMPA receptors at the level of the vagal motoneuron after winter inactivity (Figures 2–3), the amplitude fictive breath carried by vagal motoneurons should be more sensitive to inhibition of AMPA receptors.

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First, we analyzed the amplitude of the integrated motoneuron population discharge of the vagus nerve (cranial nerve X; CN X) during lung-related respiratory bursts (Figure 5A) to infer whether or not scaling of AMPA-receptors on vagal motoneurons increased the reliance of AMPA-receptors for transmitting respiratory output. An inherent limitation to measuring the amplitude of the extracellular integrated motoneuron population discharge from the cut nerve root in vitro is that only relative comparisons can be made. This precludes us from comparing absolute fictive breath amplitudes at baseline between control and winter inactivity frogs. Therefore, we rely on relative changes in burst amplitude in response to a low concentration of DNQX that did not silence respiratory bursts (4 μM) to infer differences in the reliance on AMPA-receptors for producing ‘normal,’ baseline respiratory motoneuron population amplitude in each group of frogs. In control bullfrogs, AMPA-receptor inhibition did not change the amplitude and area of the respiratory-related CN X population discharge (Figure 5B,C–D; green traces and bars), corroborating previous results in bullfrogs (Chen and Hedrick, 2008). This was not the case for bullfrogs following winter inactivity. In stark contrast, CN X population output underwent a reduction in amplitude and area during exposure to AMPA-receptor blockade (Figure 5B,C–D, blue traces and bars) and tended to recover toward baseline values following ~1 hr of DNQX washout (Figure 5—figure supplement 1). These results indicate that full transmission of respiratory CPG activity to vagal motoneurons requires an increase in synaptic strength following winter inactivity. With respect to fictive breathing frequency, brainstem-spinal cord preparations from winter inactivity bullfrogs produced an elevated number of respiratory bursts at baseline compared to control bullfrogs (Figure 5B & E₂). During exposure of the brainstem-spinal cord preparations to a low concentration of DNQX, both control and winter inactivity bullfrogs decreased fictive-breathing frequency by ~80% (Figure 5 E1), suggesting AMPA receptor-sensitive components of the respiratory rhythm generator did not differ. Collectively, our findings imply that up-scaling of AMPA-glutamate receptors on vagal motoneurons preserves respiratory motor output because, in its absence (i.e., AMPA-receptor inhibition), the amplitude of vagal motoneuron population discharge was approximately 40% less than baseline on average, while respiratory motor amplitude was unaffected by the same concentration of AMPA receptor blocker in control bullfrogs.

The mechanisms underlying how stable neuronal function emanates from inherently unstable components have been of intense interest to neuroscientists for over two decades (Abbott and LeMasson, 1993; Siegel et al., 1994; Turrigiano et al., 1994). There has been an explosion of mechanisms - postsynaptic, presynaptic, cell-autonomous, non-neuronal, etc - generated by in vitro, in vivo, and in silico approaches that paint an immensely complex picture of the genesis and maintenance of stable neuronal function (O'Leary et al., 2014; Schulz and Lane, 2017; Stellwagen and Malenka, 2006; Turrigiano, 2012). A sobering reality is that only a few studies experimentally relate mechanisms of homeostatic plasticity to the stabilization of behaviorally relevant circuit function [e.g. (Gonzalez-Islas and Wenner, 2006; Knogler et al., 2010; Lane et al., 2016). Of such studies, most opt for tractability at the expense of reality by using drastic experimental perturbations that most organisms will never experience. Although these mechanistic studies are critical, it is worth considering how animals experiencing analogous situations utilize those mechanisms for survival in their environments. After all, it is the animal in its natural environmental which natural selection acts upon.

Because respiratory motor output of bullfrogs is silent under submerged-overwinter conditions (Santin and Hartzler, 2017), they provided an ideal platform to test whether mechanisms that stabilize neuronal output over chronic time scales respond to prolonged inactivity experienced normally by an animal. In a sense, this serves as a natural analogue for experiments that dramatically reduce neural activity via pharmacological tools or injury because bullfrogs do not use neural mechanisms of ventilatory control for potentially several months during overwintering submergence. We found that respiratory vagal motoneurons underwent up-scaling of excitatory synapses in response to winter inactivity. This result is intriguing because up-scaling commonly occurs in response to experimental decreases in neuronal activity and/or synaptic transmission (Fong et al., 2015; Lambo and Turrigiano, 2013; Turrigiano et al., 1998), indicating that up-scaling of AMPA-receptors in response to inactivity-related stimuli is a physiologically relevant response in an animal normally experiencing extremely low levels of neuronal activity. Strikingly, we also observed that vagal motoneuron population output driven by respiratory CPG activity was ~40% less than baseline when we, presumably, blocked the effects of scaling with a sub-saturating concentration of AMPA-receptor antagonist after winter inactivity. This contrasts with no effect of AMPA-receptor block on respiratory motor amplitude in control bullfrogs (Figure 5). Given that amplitude of population motoneuron firing corresponds with drive to respiratory muscles in vivo (Sakakibara, 1984b), up-scaling seems to ensure adequate motor output to critical breathing muscles. Since lung ventilation satisfies gas exchange requirements immediately after winter inactivity in intact, unrestrained bullfrogs (Santin and Hartzler, 2016a), our findings suggest that scaling of AMPA receptors on respiratory motoneurons may contribute to normal ventilation after months without lung breathing.

Several lines of evidence suggest this is the case. First, we observed an increase in mEPSC amplitude and charge transfer without changes in neuronal input resistance and mEPSC rise time, indicating that increased function of AMPA-glutamate receptors, rather than differences in electrotonic properties, explain the increase in mEPSC amplitude (Figure 2) (Han and Stevens, 2009). Whether post-synaptic enhancement of AMPA-receptor function (via elevated expression or post-translational modification) or a presynaptic synaptic mechanism (Liu et al., 1999) causes the larger mEPSC remains an open question. Second, ranked ordered mEPSCs from control and winter inactivity distributions were well-fit by a linear regression, a hallmark of synaptic scaling, instead of an exponential curve, which describes the rank order relationship following LTP (Figure 3A) (Gainey et al., 2009). Third, dividing the winter inactivity mEPSC amplitude distribution by the scaling factor obtained from the linear fit of ranked order mEPSC amplitudes produced a ‘down-scaled’ distribution that overlays the control mEPSC amplitude distribution (Figure 3B) (Turrigiano et al., 1998). Points two and three imply that AMPA-receptors scaled slowly and globally throughout the duration of winter inactivity, rather than rapidly through LTP-like mechanisms during the time between removal of the frog from the simulated winter environment and ~3 hr later when the recordings were made. However, the timing of induction and completion of up-scaling in vagal respiratory motoneurons during the course of the 2-month period without breathing is unresolved. Finally, increased synaptic strength enhanced the contribution of AMPA-receptors for producing respiratory motoneuron discharge. Unlike control bullfrogs, vagal motoneurons receiving respiratory CPG input reduced motoneuron population amplitude by ~40% after winter inactivity upon exposure to a low concentration of AMPA-receptor antagonist (Figure 5). Consistent with a compensatory or homeostatic response, this result suggests that respiratory output leaving the brainstem through the vagus nerve would have been absolutely smaller in bullfrogs after winter inactivity if scaling did not occur. Since motoneuron population discharge increases proportionally with outflow to respiratory muscles in vivo (Sakakibara, 1984b), we collectively interpret our results to mean that scaling of AMPA-receptors on respiratory motoneurons is part of a strategy used to preserve respiratory motor output that produces appropriate lung breathing after winter inactivity.

Although the amplitude of vagal motoneuron output became increasingly reliant on AMPA-receptors following winter inactivity, upregulation of AMPA-receptors probably does not underlie the facilitation of fictive-breathing frequency that we observed (Figure 5E present study, Santin and Hartzler, 2016b). This implies that respiratory rhythm generating circuits use distinct mechanisms of compensation to enhance the frequency of respiratory-related discharge following winter inactivity. Although the mechanisms are presently unknown, several, potentially interacting, processes could underlie enhanced burst frequency in vitro after winter inactivity. These mechanisms may include, but are not limited to (1) decreasing the inhibitory GABA/glycinergic tone within rhythm generating circuits (Straus et al., 2000), (2) altering the neuromodulatory state of rhythm generating circuits, and (3) decreasing the O₂ sensitivity of the respiratory control system that may act to tonically depress fictive lung bursts (Santin and Hartzler, 2016b; Winmill et al., 2005). Although the mechanisms leading to enhanced fictive lung burst frequency remain untested, it is apparent that a rich repertoire of compensatory mechanisms may ensure reliable action of the different components of the respiratory control system after months of inactivity.

Exciting questions remain for homeostatic plasticity that can only be understood using animals in natural environments; mainly, how do such compensatory mechanisms operate in natural contexts? An inability to understand principles linking induction and maintenance of these compensatory mechanisms with normal physiological challenges makes it difficult to imagine that these mechanisms can be extrapolated and understood in the context of human pathologies for which they may play a role (Beck and Yaari, 2008; D'Amico et al., 2014; Rajman et al., 2017). Recently, factors associated with physiological state (e.g. sleep-wake) have been suggested to induce synaptic scaling (Diering et al., 2017) implying that neuronal circuits in intact animals use homeostatic and compensatory mechanisms that extend beyond the simple paradigms in which they are commonly studied. The respiratory motor network of bullfrogs following winter inactivity provides a tractable and ecologically relevant model to address major questions regarding preservation of nervous system output. For example, how do neurons ‘know’ when to scale their synapses during physiological challenges (or even when to stop scaling), do specific or multiple interacting stimuli trigger synaptic scaling during natural perturbations in vivo, and why do different components of a behavioral control system apparently use distinct compensatory mechanisms? Only considering mechanisms in the context of realistic challenges encountered by the animals in which they exist can the plethora of mechanisms underlying brain stability be brought to life.

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Author details

1. Joseph M Santin

   1. Division of Biological Sciences, University of Missouri-Columbia, Columbia, United States
   2. Department of Biological Sciences, Wright State University, Dayton, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   santinj@missouri.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1308-623X

2. Mauricio Vallejo

   Department of Biological Sciences, Wright State University, Dayton, United States

   Contribution

   Data curation, Formal analysis, Writing—review and editing

   Competing interests

   No competing interests declared

3. Lynn K Hartzler

   Department of Biological Sciences, Wright State University, Dayton, United States

   Contribution

   Supervision, Investigation, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2355-0174

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