The initial aim of the experiments was to investigate the occurrence of CRDPs in hindlimb motoneurons of decerebrates such as were previously reported to occur under anesthesia (Kirkwood et al., 2002; Ford and Kirkwood, 2006), concentrating in particular on their voltage dependency. Our monitor nerve discharge was therefore that of the phrenic (or, in one instance, an external intercostal nerve in lieu of the phrenic). In all preparations, CRDPs were common, almost all being of the expiratory decrementing (Edec) type, as in Ford and Kirkwood (2006). In 4 out of the 7 experiments, the amplitudes of the CRDPs were relatively small, and often showing some amplification with depolarization, again similar to that previous study, although now with a higher occurrence of plateaux. When tested, no efferent discharges were present in the hindlimb nerves. In contrast, in the remaining three experiments, the respiratory drive in the hindlimb motoneurons was often strong, and additionally at some times during the experiment spontaneous LDPs were also seen. In the first of these experiments, the LDPs were only identified in the recordings *post hoc*, but in the other two experiments, the LDPs were confirmed as such by recording periodic (often flexor-extensor alternating) efferent discharges from the hindlimb nerves. These three animals were also characterized by periodic fluctuations in blood pressure, slower than the respiratory rate, with intervals between the blood pressure maxima ranging from 9 to 104 s, though at any moment during an experiment, the interval could be very variable. The mean interval was assessed in each of the computer runs where the fluctuations could be readily distinguished, the mean of these values being 39 s ± 17 SD (*n* = 71). We are taking these as Mayer waves, despite the frequencies mostly being slower than those classically recognized under this term (see Discussion). Here the Mayer waves appeared variably in the course of the recording sessions, often occurring as isolated phases of increased blood pressure on a steady background (e.g., see later in **Figures 5**, **9**). However, they also could occur, especially when the frequency was higher, as a continuously fluctuating waveform or, occasionally, as periodic dips in blood pressure from a steady high level (e.g., see later **Figure 3**).

Clinically, Mayer waves may be seen in pathological situations (e.g., in hypovolemia), but it should be noted that these three preparations were among those in apparently the best condition. One of them, which provides many of the illustrations here, was specifically noted as such in the experimental protocol, before any recordings had been made. In this animal there had been minimal blood loss during the decerebration procedure and it showed a brisk CO2 response in the phrenic nerve, including hypocapnic apnea. An overall characteristic of these three preparations was a minute-to-minute variability of state, as reflected in the patterns of the Mayer waves, CRDPs, LDPs, and nerve discharges. However, a universal observation was that whenever the Mayer wave behavior was marked then either or both of the respiratory and locomotor drives were modulated in relation to the Mayer wave cycle. It is this modulation which is the subject of this report.

As illustrated in **Figure 1** (and later in **Figure 5**), most often the phrenic nerve discharge in the inspiratory phase just preceding the blood pressure rise was particularly intense while the

phrenic nerve discharges in the following respiratory cycles were less intense than in steady state, a modulation with a similar phase difference to that shown by Preiss et al. (1975). **Figure 1** includes two different examples of the phrenic modulation,

were less intense. In **(A)** the initial increase prior to the Mayer wave is difficult to judge as it occurred at the very beginning of the recording, but the less intense discharges during the blood pressure increase are easily seen. The following three traces show **(A)** the raw ENGs from SmAB and PBSt,

> a relatively continuous variation in **Figure 1A**, and a modest increase of probably only one phrenic burst per Mayer-wave cycle in **Figure 1B**. Another variation, even closer to the behavior described by Preiss et al. (1975), was one where several respiratory

bursts were present at the start of the hypertensive phases, but were accompanied by variable bursts during the end of inspiration and later into subsequent respiratory cycles. Vertical dotted lines indicate the end of

inspiration.

cycles showed an elevated phrenic discharge starting just before the blood pressure rise. An illustration of this was included in the Supplementary data of Enríquez Denton et al. (2012). **Figure 1** also illustrates one of the typical patterns of nerve discharges associated with the occurrence of Mayer waves, a co-activation of flexors and extensors. Most often, as in **Figure 1A**, this generalized nerve discharge occurred in the post-inspiratory phase, briefly after the end of the phrenic nerve discharge (Richter, 1982). However, with the first Mayer wave in **Figure 1B** the co-activation actually started during late inspiration and it was repeated a few times during the period of elevated blood pressure before falling silent. At the second Mayer wave in **Figure 1B** there was only a very brief and weak co-activation during inspiration, while the major discharge occurred, as usual, in the post-inspiratory phase. The two examples in **Figure 1** were typical of individual Mayer wave cycles in each of those two preparations in the particular states pertaining at the time (see later for a description of state variation). We describe below some of the variations seen in the other states.

In some cases the post-inspiratory co-activation of flexors and extensors occurred for each respiratory cycle for prolonged periods when Mayer waves were weak or absent, as seen in **Figure 2**.

The occurrence of Mayer waves was also frequently related to the initiation of short-lasting episodes of fictive locomotion. One such case is illustrated in **Figure 3A** in which locomotor-like activity is seen during the high-pressure periods in three Mayer wave cycles. At a faster time base in **Figure 3B** it is seen that the knee flexors PBSt alternated with the activity in the hip extensor SmAB and ankle extensor GS. However, the antagonist ankle flexor TA nerve activity coincided with the GS activity in this particular example (see further in the Discussion).

## **INTERFERENCE BETWEEN MAYER WAVE-RELATED HINDLIMB NERVE ACTIVITY AND SPONTANEOUS FICTIVE LOCOMOTION**

One essential question emerging from the results in the preceding section is related to the identity of the spinal circuits mediating the respiratory-related discharge in the hindlimb muscle nerves. Are they part of the spinal CPG for locomotion or unrelated to that network? In the un-anesthetized decerebrate preparation locomotor-like activity sometimes develops spontaneously (Frigon and Gossard, 2009), as was the case for the three preparations, whose behavior we describe here. In these preparations we noted various examples of perturbations (resetting) and entrainment of the spontaneous locomotor rhythm by the central respiratory activity as monitored by the phrenic nerve discharge, and where the intracellular motoneuron recordings gave us particular insights.

The motoneuron recordings shown in **Figure 4** illustrate two aspects of the interaction between the respiratory and locomotor rhythms. In **Figure 4A** (a PBSt motoneuron) it can be seen firstly that, during the periods when the blood pressure was rising and maintained at a high level (right-hand side of the figure), every discharge in the phrenic nerve (marked by open arrowheads on top of the phrenic nerve ENG) was clearly related to a short interruption of PBSt nerve discharges (partly also seen in the SmAB nerve) together with a striking decrease of the flexor-related LDPs (filled arrows). During such a period, the PBSt discharge was exaggerated in the immediate post-inspiratory phase, as was the depolarization in the motoneuron. Secondly, in the relatively short periods of reduced blood pressure (and also during the decay phase of the preceding Mayer wave), the LDPs were not just interrupted or modulated, but are replaced by a post-inspiratory CRDP, the third cycle of which (immediately before the LDPs commence) appears to involve a plateau potential. A somewhat

similar sequence is seen in **Figure 4B**, where the respiratory and locomotor rhythms again alternated according to the phase of the Mayer wave cycle. This can be seen in the nerve discharges, but is particularly clear in the (GS) motoneuron recording in this instance. At the beginning of the recording (10–20 s, during the decay-phase of the preceding Mayer wave and the period with low blood pressure), the nerve activity was dominated by the respiratory drive then followed by locomotor activity, corresponding to the rising blood pressure. The CRDP in this instance is dominated by a post-inspiratory inhibition (together with a PBSt discharge). This is significant, because it suggests that, at least in some instances when only a single (post-inspiratory) phase of the respiratory drive is evident in the hindlimb discharges, important components of the interneuron circuitry involving reciprocal inhibition appropriate to locomotion may nevertheless be recruited. These examples strongly suggest that the respiratory drive to the motoneurons not only interferes with, but may be mediated by, the spinal locomotor CPG circuits.

In **Figure 5A** there is an example of ongoing locomotor activity (seen as LDPs in the recorded SmAB motoneuron). In the middle of the recorded period there is a large inspiratory discharge (box) followed by a Mayer wave as in **Figure 1**. The phrenic nerve recording and the intracellular drive potential are shown at a faster time base in **Figure 5B**. In **Figure 5C** we show the variation—or rather stability—of the locomotor cycles, with the exception of cycles 10 and 11 marked below the motoneuron recording. It is obvious that in relation to the large phrenic nerve discharge just before the BP increase there is a very short cycle (i.e., the depolarising phase starts earlier than expected) followed by a very long duration for the next cycle. There is resetting of the locomotor rhythm here (the sum of cycles 10 and 11 is longer than any other pair of adjacent cycles), but it is relatively subtle: the timing of the rhythm recovered after a very brief interruption and it is not clear whether the respiratory input really caused an interference with the locomotor CPG rhythm-generator, or whether the modulation coinciding with the Mayer wave event simply slowed the locomotor rhythm and the large depolarisation reflected convergence at motoneuronal level.

The data of **Figure 5** come from a long period (about 3 h) in one cat, where a continuous locomotor rhythm was present (see later section). Despite the presence of isolated, relatively large Mayer waves as in **Figure 5**, there were also periods of minimal Mayer wave activity. We therefore looked carefully at these periods for indications of influence of the respiratory rhythm on the locomotor rhythm independent of Mayer wave occurrence. **Figure 6** illustrates one of these periods. In A the raw records are included from a part (33 s) of the 100 s long recording (in all 17 phrenic nerve discharge cycles; only few of them are illustrated in **Figure 6A**). Even from these raw records it can be seen that there is a tendency for the phrenic discharge to be followed closely by a flexor discharge (illustrated for PBSt), but there were also examples where this was not the case (e.g., the phrenic nerve discharges marked by asterisks in the upper rectified trace in the period illustrated in **Figure 6A**). The tendency of a post-inspiratory flexor discharge was formally tested for all 17 respiratory cycles. In **Figure 6B1** we show the discharges of the PBSt nerve from the recording shown in A, averaged in relation to the offset of the phrenic nerve burst, which demonstrated

**FIGURE 4 | Expanded records of Mayer wave-initiated locomotor-like activity during intracellular recording from two motoneurons. (A)** From a PBST motoneuron. The largest bursts in the PBSt nerve occurred in the post-inspiratory phase of respiration, corresponding to the largest depolarizations of the LDP in a PBSt motoneuron (asterisks for both). These depolarizations often elicited spikes (truncated). During inspiration, indicated by open arrowheads above the phrenic recording (whose discharge appears weak, most likely a recording problem), there was a decreased amplitude of the LDP (arrows). Between the episodes of locomotor-like activity, the respiratory drive was evident as a CRDP in the PBSt motoneuron, consisting of rather variable depolarization in post-inspiration (arrowheads), and with a corresponding weak discharge, just detectable in the PBSt nerve. The prolonged depolarization visible in the intracellular record following the third arrowhead corresponds to

the start of the locomotor episode and probably involved a plateau potential. Same nerve recordings as in **Figure 3**. **(B)** Similar behavior seen in a recording from the next motoneuron (GS) penetrated in the same preparation. Again, a CRDP is seen in the intracellular recording, in alternation with an LDP, according to the phase of the Mayer-wave. The GS nerve was silent at this time (not shown), but the apparent strong post-inspiratory inhibition in the motoneuron shows the alternation between GS and PBSt for both the CRDP and LDP, similar to that between TA and PBSt during the locomotor episodes. TA was silent during the respiratory episodes, but alternated with PBSt during the locomotor episodes. It is possible that persistent inward current contributed to the depolarizations in this cell, but periods of de- and hyperpolarizations (not shown) indicated that strong phasic inhibition of a tonic background excitation could be sufficient to explain the CRDP/LDP in this instance.

an apparent entrainment with a post-inspiratory flexor burst, and 4–5 locomotor cycles for each respiratory cycle. Note that the amplitude of the averaged waveform is about 60% of that of the raw integrated rectified PBSt discharge, thus indicating a rather strong phase-coupling. These features (including the same phase relation of the average to the phrenic discharge) were preserved, when this period of 17 cycles was split into two separate periods (7 and 9 cycles, data not shown). The

entrainment appears strongest when averaged in relation to the end of the phrenic discharge, most likely because the predominant excitatory event occurred in the post-inspiratory phase and the duration of the phrenic discharge was variable (cf. Richter, 1982). However, the averaged PBSt discharge is clearly visible also when triggered on the onset of the phrenic discharge (**Figure 6B2**).

The animals were paralyzed and artificially ventilated at a rather high frequency and low volume, with pneumothorax (cf. Methods). Nevertheless, it would have been possible that activation of thoracic, abdominal or vagal mechanoreceptors could have entrained the locomotor (and/or respiratory) rhythm. However, when the PBSt activity was averaged in relation to the pump (offset of inflation) there was absolutely no sign of entrainment (**Figure 6B3**). Therefore, we conclude that in this case there is a strong entrainment of the locomotor rhythm by the intrinsic respiratory rhythm.

One other such period (20 phrenic cycles) showed the same effect (the same phase), but in five other periods analyzed, this was not really the case. The amplitude of the averaged PBSt ENG was very small suggesting the lack of a strong phase-locking, and thus a lack of evidence for entrainment. Overall, therefore, this long period of continuous locomotor discharge represented a wide spectrum of effects, with examples of a strong coupling and more often an independence between the two rhythm generators.

## **VOLTAGE-DEPENDENT AMPLIFICATION OF LDPs, MUSCLE STRETCH EVOKED EPSPs, AND CRDPs**

A general finding in these experiments was that all of these drive potentials showed a strong voltage-dependent amplification in the voltage region where recruitment of PICs would be expected to contribute to an enhancement of their amplitudes. Such measurements are not always easy to obtain since reciprocal inhibition (through different inhibitory interneurons in between the excitatory periods) also will increase the peak-to-peak amplitude at increasingly depolarized levels. Furthermore, the membrane resistance is reduced at depolarized levels (both by opening of PIC channels and K+ channels). Nevertheless, at least qualitative evidence for such amplification was commonly obtained. **Figure 7A** shows the voltage-dependent amplification of an LDP in a SmAB motoneuron in confirmation of Brownstone et al. (1994). This was demonstrated in 16 out of 16 motoneurons in the present experiments, although a sizeable inhibitory component was suspected in five of these). The decrease of the LDPs is best seen during the hyperpolarizing (descending) phase of the triangular current injection. During the peak of current injection the spikes inactivated and there was an obvious shunting. In **Figure 7B** (confirming Bennett et al., 1998) there is a similar potential-dependency of the EPSPs evoked by sinusoidal triceps surae stretches (in a GS motoneuron).

The voltage-dependent amplification of CDRPs (demonstrated in 12 out of 12 motoneurons) was sometimes very dramatic. In **Figure 8** we illustrate how a very small Edec CRDP (in a GS motoneuron) was enhanced as the holding potential was increased from −82 to −75 mV and up to −62 mV. At the last level the depolarising phase triggered plateau potentials that usually fell off soon after initiation, and were fully terminated by a subsequent presumed inhibitory phase. These occurred in an all-or nothing fashion. The first two inspiratory discharges after the last depolarizing current step were not followed by plateaux, then the first plateau was not related to the inspiratory discharge, but appeared to be triggered spontaneously. At the very end of the trace the plateau potential was sustained for the whole respiratory cycle. In order to better visualize the potential-dependency we have averaged the intracellular recordings at the three different levels of current injection (−4 nA, +2 nA, and +6 nA) with reference to the beginning of inspiration (**Figure 8B**). The averages here show first, the presumed inhibitory component during inspiration, increasing, as expected, with depolarization, then, at +2 nA, a small, late depolarization occurred about 0.5 s into expiration.

**FIGURE 7 | Voltage dependent amplification of LDPs in a SmAB motoneuron and stretch-evoked Ia excitation in a GS motoneuron. (A)** Recording from a SmAB motoneuron during spontaneous locomotor-like activity. The traces show the LDPs (top), the current injection (middle) and the SmAB ENG (bottom).The voltage dependent amplification of the LDPs (those between the arrowheads) are shown at the descending phase of a triangular current pulse. **(B)** Recordings from a GS motoneuron. The traces show the stretch-evoked EPSPs (top), the current injection (middle) and the sinusoidal muscle stretch of triceps surae (bottom). The arrowheads indicate the region where the amplification was present. At more depolarized levels the amplitude of the EPSP was smaller because of shunting (increased conductance), while the amplitude decreased at more hyperpolarized levels due to the voltage dependent amplification of the EPSP.

At +6 nA, the plateau-like depolarizations occurred at variable times between 0 and 0.5 s into expiration, as indicated by their initial spike-like components, which are clearly preserved in the average. However, because of this variability, the amplitude of the average plateau is attenuated as compared with the individual events.

## **EVIDENCE FOR CHANGES IN INTRINSIC MOTONEURON PROPERTIES IN RELATION TO MAYER WAVES**

There is much evidence for Mayer waves reflecting an increased sympathetic drive at the postganglionic level (see Discussion). However, there is no direct evidence for a simultaneously

The traces show a CRDP in a Tib motoneuron (top) during step-wise current injection (middle) and the T5 external intercostal ENG activity (bottom). The CRDP becomes larger at at more depolarized membrane potential levels. **(B)** The averaged CRDPs from **(A)**. The averages were performed separately at the three membrane potential levels

inspiration. Note in the average the increased amplitude of the CRDP and the variable times of occurrence for the plateau-like events at +6 nA, which produces a relative attenuation in the averaged signal for this level (*n* = 7, 6, 7, respectively from low to high potential levels). Spikes truncated.

increased monoaminergic drive to the spinal cord, and we have not aimed to obtain direct evidence for such a drive in the current experiments (see further in Discussion). However, we have looked for signs of changes in intrinsic properties in the motoneurons during Mayer waves that are compatible with an increased monoaminergic drive onto them, promoting PICs and plateau properties. **Figure 9A** demonstrates how triangular current injections triggered plateau potentials (with a couple of initial spikes). The illustration shows a plateau potential that was initiated and terminated with lower current injection and at a more hyperpolarized membrane potential during the Mayer wave than during the first triangular current injection (no Mayer

wave). The longer-lasting plateau during the descending phase during the Mayer wave is particularly striking, but from amplified records we could also confirm that the plateau was initiated at a 2.4 mV more hyperpolarized level (and with 3 nA less depolarizing current injection) than during the first trial before the onset of the Mayer wave. In another illustration, in **Figure 9B** it is noted that a spontaneous plateau potential was triggered "off-cycle," toward the end of the phase of increased inspiratory

CRDP recorded in a TIB MN. In this case the Mayer wave is inferred by the

discharges (filled arrow) during the putative Mayer wave (in this case we did not record the blood pressure, but the large intercostal nerve discharges followed by a number of less intensive discharges (marked by an open arrow) at the following respiratory cycles were taken as an indirect indication of the Mayer wave cycle). This same sequence was repeated 5 times in this particular cell. In another motoneuron shown in **Figure 9C** it may be seen how the small potential fluctuations did not trigger a plateau potential

of these triggered both an intense firing and a plateau potential.

at normal blood pressure levels, but they did so when occurring at the very beginning of a Mayer wave. There were several examples of this kind, but as most of the Mayer waves appeared at irregular intervals a systematic investigation turned out to be difficult. Nevertheless, the present observations are highly suggestive of a neuromodulation that enhances the plateau-properties of motoneurons in relation to Mayer waves.

### **EVOLUTION OF THE GENERAL STATE OF EACH PREPARATION**

In the preceding sections we have described a variety of different patterns of motor output. These did not occur at random, but each of the three preparations here showed a systematic change of state during the course of the experiment, though different for each preparation. Each of the observations reported in the preceding sections is only a snapshot, but each is nevertheless representative of longer recordings in that cell, and of the state. The recordings covered periods of 11, 7, and 9 h, respectively for the three preparations, and consisted of individual motoneuron (MN) recordings lasting from about 1 to 40 min. The states of each preparation can be summarized as follows.

## *Cat 1*

Initial hindlimb nerve recordings were made in the absence of Mayer waves and showed PBSt discharges during post-Inspiration similar to those in **Figure 2** (although that illustration was actually derived from Cat 3). The initial 4 motoneurons yielded an uncertain picture as the phrenic nerve recording gradually failed. By the time an intercostal nerve had been prepared and intracellular recording resumed, Mayer waves were present and small amplitude presumed LDPs, amplified by depolarization, were detectable, sometimes alternating with CRDPs, according to the Mayer wave phase, which itself was clear from the intercostal nerve recording (as in **Figure 9B**). The amplitude of the Mayer waves was unknown (the blood pressure recording was not acquired by computer in this experiment), but was large enough by the time MN 10 was recorded to be noted in the protocol as an unusually large blood pressure variation being present. The CRDPs then became the dominant feature in the remaining motoneurons (up to MN 22). These CRDPs were strongly amplified by depolarization, involved plateau potentials, and were modulated in phase with the Mayer waves (**Figures 8**, **9B**).

## *Cat 2*

Initial nerve hindlimb recordings were made during strong Mayer waves (amplitudes up to 45 mm Hg) with respiratory phased discharges in the nerves strongly modulated in phase with the Mayer waves (**Figure 1A**). During the early intracellular recordings, Mayer waves were initially absent, then returned (MNs 5, 6), then faded again (MNs 7–9). No LDPs and only very small CRDPS were present. The baseline blood pressure was steady at around 120/80. The phrenic discharge was only modulated when the Mayer wave amplitude was as high as 20 mm Hg. Between MNs 9 and 10 the blood pressure fell, but was restored by a single dose of Effortil (0.5 mg/kg). Following this, a strong locomotor drive became apparent, which persisted during the remaining 4 (relatively long) motoneuron recordings of this experiment. The Mayer wave amplitudes were smaller than in the initial runs (15–20 mm Hg) but nevertheless strong modulation of the motor discharges was apparent in phase with the Mayer waves (**Figures 3**, **4**). The Mayer waves gradually decreased in amplitude, but the modulation of the locomotor and respiratory discharges related to them persisted with amplitudes below 10 mm Hg.

## *Cat 3*

This involved the largest number of motoneuron recordings (MN1–MN33). For the first 14 motoneuron recordings, only low amplitude Mayer waves were present, only low amplitude CRDPs and no LDPs. A weak respiratory-phased discharge was present in SmAB nerve in the one recording tested in this period. Modulation of the CRDPs and of the phrenic discharges in relation to the Mayer waves was only seen toward the latter part of this period (MNs 9–14) and only for larger amplitude Mayer waves (10–15 mm Hg). Then, for MNs 16–26, continuous LDPs were present, together with continuous locomotor discharges in the hindlimb nerves, as in **Figures 5**, **6**. Mayer waves were only sometimes present, generally of low amplitude (≤15 mm Hg), and these were not accompanied by any obvious modulation of the motor output. However, occasional isolated large amplitude Mayer wave events (up to 40 mm Hg) also occurred, accompanied by resetting of the locomotor rhythm with regard to the respiratory cycle (**Figure 5**). For MNs 27–31, the Mayer waves became stronger and more regular, being accompanied first by a continuous respiratory drive in the hindlimb nerves in the periods between mediumamplitude Mayer wave events (**Figure 2**), then by the strongly modulated short (locomotor?) repetitive bursts synchronized to respiration and to large amplitude (45 mm Hg) Mayer waves (**Figure 1B**). For the last two motoneuron recordings, the Mayer wave amplitude decreased, as did the modulation of the locomotor discharges.

Overall, therefore, although not designed for this purpose, the structure of the experiments, which consisted of repeated measurements over many hours, revealed two or three different time scales of spontaneous modulatory variation. The first one, varying over hours to minutes, seemed to consist of the extent to which locomotor drives were present at all, a respiratory drive being present throughout (as it was in the four experiments not showing Mayer waves). The second one, varying over minutes to seconds determined, in phase with the Mayer waves, which of the two rhythms was transmitted to the motoneurones. Finally, the Mayer waves themselves waxed and waned independently of the locomotor drive, but over a similar time scale.

## **DISCUSSION**

## **RELATIONS BETWEEN THE NEURONAL NETWORKS OF RESPIRATION AND LOCOMOTION AT LUMBAR LEVEL**

In this report we have demonstrated that at spinal level there are intricate interrelations between the networks mediating a respiratory control of lower limb muscle activity and locomotor activity. In the decerebrate, unanesthetized and paralyzed preparation which we have used, the activation of these interneuronal circuits—and their interrelation—varied substantially with the spontaneous occurrence of periods of increased blood pressure, i.e., the so called Mayer waves. During these periods the intrinsic behavior of motoneuron properties changed as might be expected if an increased monoaminergic drive to the spinal cord occurred in phase with the presumed sympathetic activity during the Mayer waves (cf. the examples in **Figure 9**, see below). It is well-established that monoamines are important transmitters in activating the spinal network underlying locomotor activity, the spinal locomotor CPG, as first demonstrated for the cat (administration of l-DOPA: Jankowska et al., 1967a,b; Grillner and Zangger, 1979, or the α<sup>2</sup> agonist clonidine, Forssberg and Grillner, 1973; Barbeau et al., 1987; Kiehn et al., 1992; Chau et al., 1998; Noga et al., 2006, 2007). In addition monoamines (NA; DA and 5-HT) have been used to activate spinal locomotion in the rabbit (Viala et al., 1979) and the marmoset monkey (Fedirchuk et al., 1998) as well as in adult rats (Iles and Nicolopoulos-Stounaras, 1996) and mice (Meehan et al., 2012). Monoamines, often in combination with NMDA receptor agonists, also activate the spinal CPG in the neonatal rat and mouse, (reviews; Schmidt and Jordan, 2000; Clarac et al., 2004; Miles and Sillar, 2011). Readers should also consult other articles in this Research Topic.

In general, the locomotor pattern evoked by the pharmacological cocktail is characterized by alternating flexor and extensor bursts, and by alternation between the two hindlimbs. However, at the start of the bursting activity it has been frequently noted that flexors and extensors were initially co-activated, as were sometimes the two limbs, and that the regularly alternating pattern only subsequently developed during the course of the recording session (Fedirchuk et al., 1998 in the marmoset monkey; Meehan et al., 2012 in the mouse; and more infrequently in the cat preparation, unpublished observation from Hultborn's laboratory). These observations are relevant for the interpretation of the pattern of co-activation of flexors and extensors in the post-inspiratory phase (**Figures 1**, **2**). One interpretation would be that the interneurons mediating such co-contraction are different from the locomotor circuits, but an alternative explanation would be that the respiratory drive is activating parts of the locomotor network in a functional configuration which causes a co-activation. As support for the latter explanation the prolonged locomotor periods in **Figures 3**, **4** actually display a co-activation of the ankle extensors (GS) and flexors (TA), although flexors and extensors otherwise were alternating. Even more convincing evidence for the respiratory drive working onto—and at least partly through—the locomotor circuits are the inhibitory components of the CRDP in **Figure 4B**, together with the possible resetting (**Figure 5**) and certain entrainment (**Figure 6**) of the locomotor rhythm by the respiratory one.

A correlation between actual motor activity and respiration has long been recognized, and is of course related to increased demand for oxygen and removal of CO2. The regulation of the increased ventilation is complex and involves both feed-forward and feed-back mechanisms. It has been studied in humans (Krogh and Lindhard, 1913; Asmussen, 1983; Haouzi, 2006), as well as during the initiation of locomotion in the decerebrate cat (DiMarco et al., 1983). In relation to locomotion, several groups have established an entrainment of the respiratory rhythm by the locomotor activity (Kawahara et al., 1989), which to a large extent seems to originate from limb proprioceptive inputs (Potts et al., 2005; Giraudin et al., 2012), but there is also evidence for a central entrainment of the respiratory pattern by the spinal locomotor circuits (Perségol et al., 1988; Le Gal et al., 2014). The interactions between these two CPGs, the medullary respiratory one and the spinal locomotor one have been described at length in a series of papers from Viala and her colleagues, including also the likely cervical spinal respiratory oscillator, which is not of concern here. In nearly all of these papers the focus is on the dominance of the locomotor over the respiratory CPG, though in two publications from this group (Viala, 1986; Perségol et al., 1988), the authors allowed for the reverse to occur. In these papers (both including fictive activations) the authors describe how the dominance of one rhythm over the other can be varied. The present observations, which show an entrainment of the locomotor circuits by the respiratory rhythm, are closest to their data in the circumstances of a raised level of CO2, which was also the circumstance of most of our observations. However, their preparations were different from ours: rabbits with, for the critical observations, a very caudal decerebration and locomotion induced pharmacologically. We suggest the dominance of the respiratory over the locomotor CPG was even stronger for our observations, because the high level of CO2 was combined with only spontaneous locomotion. Some of our recordings, where a locomotor pattern alternated with a respiratory one were also remarkably similar to one previous publication in the cat (Figure 5B in Romaniuk et al., 1994). It may be proposed that by stimulating within the more caudal part of the "locomotor strip" (in the medulla) Romaniuk and his colleagues also gave only a relatively weak drive for locomotion. The only difference between their result and ours is that their forelimb nerve discharge showed an inspiratory rather than postinspiratory pattern when the locomotor pattern did not prevail. However, this is quite compatible with previous observations of respiratory drives recorded intracellularly in forelimb (Enríquez Denton et al., 2012) or hindlimb (Ford and Kirkwood, 2006) motoneurons. By showing the linkage between the two CPGs our present observations also help to explain the many various earlier observations of respiratory influences on limb functions. Meyer-Lohman (1974) can be consulted for an early description of post-inspiratory effects in hindlimb motoneurons, and for even earlier references of respiratory effects.

The question of what function is served by these interactions remains. In addition to variable interactions between the central rhythm generators for spinal locomotor and respiratory control in the adult preparations, the coactivation has also been described in the fetal (Greer et al., 1992) and in the neonatal rat preparations (Morin and Viala, 2002). The coactivation of these CPGs and their variable coupling likely represents an "open-loop" operation of these networks with reduced "functional" control, as both feed-forward and feed-back mechanisms are depressed in the preparations used for studying them. In other words, the inter/descending/propriospinal-neuron networks coupling these two systems are hard-wired in the spinal-brainstem circuits very early during development, and they become very much under the control of sensory feedback and feed-forward signaling from the brain as the animals mature. Under such control, the role of the interactions could be many and varied (see Discussion in Schomburg et al., 2003).

For interactions in the direction of locomotion to respiration, the function is the most obvious, and has been much discussed in terms of respiratory efficiency, or for minimum work in general, especially in situations such as galloping or, for birds, flying (Boggs, 2002). In the opposite direction, as suggested here, the function is less obvious. In this context, it should be remembered that the respiratory CPG, or the neurons within it, can be readily configured for other functions, which include vomiting, coughing, defecation, perhaps even mating (for references see Kirkwood and Ford, 2004). Thus, the influences we are reporting from decerebrate preparations may reflect the operation of connections evolved for motor acts other than respiration, and where the roles of the limbs may be more essential. These roles may also be different for the fore- and the hindlimbs. For the latter the connections may serve the functions just suggested, for the former they may serve accessory inspiratory actions. A large number of possible functions (largely still speculative) for the post-inspiratory (or Edec) pattern of excitation in hindlimb motoneurons were discussed by Ford and Kirkwood (2006).

## **DO MAYER WAVES REFLECT AN INCREASED DESCENDING MONOAMINERGIC DRIVE TO THE SPINAL CORD?**

First we will summarize some facts and questions about the so-called Mayer waves. Slow oscillations (6–9 cycles/min) in arterial pressure were described by Mayer in 1876 in anesthetized rabbits (Mayer, 1876). The review by Julien (2006) describes that the dominant frequency varies between species (∼0.1 Hz in humans; ∼0.3 Hz in rabbits and ∼0.4 Hz in rats and mice), and also that the value depends on the experimental circumstances such as anesthesia, body position and circulatory conditions. In experimental animals and in humans, simultaneous recordings of low frequency increases in arterial blood pressure (at much slower frequency than the respiratory movements themselves) and efferent sympathetic activity have revealed a strong correlation (see review by Julien, 2006). Nevertheless, the origin of these oscillations has been the subject of much experimental work and discussion, mainly focusing on two possible explanations which are not mutually exclusive; (1) the pacemaker theory assuming a central oscillator (in the brain stem) for the genesis of Mayer waves, and (2) the reflex theory emphasizing the baroreflex loop. A large number of experimental studies (reviewed by Julien, 2006) support both hypotheses. In animal experiments the baroreceptor loop can be easily opened either by surgical denervation or by pharmacological means, and this intervention certainly reduces the Mayer wave activity. On the other hand there are observations on slow waves of sympathetic nerve activity even when the blood pressure is clamped and the baroreceptor loop opened, thus emphasizing the presence of a central pacemaker (Preiss and Polosa, 1974; Preiss et al., 1975). As pointed out by Malpas (2002), evidence such as baroreceptor denervation reducing Mayer wave activity is very weak as to the baroreceptor role in an oscillating feedback loop, it only shows that these receptors provide a facilitatory input. However, experiments which show that the activity survives the opening of such a loop, such as those in Preiss and Polosa (1974), provide very strong evidence that Mayer waves can be generated purely centrally. Modern reviewers are dismissive of this evidence, because the Mayer wave frequency in those experiments was much lower and the amplitudes of the pressure variations were much higher than those usually now considered as Mayer waves, and also because Preiss and Polosa (1974) induced these waves by severe hypovolemia. In terms of amplitude and frequency of the blood pressure variation, our experiments are close to those of Preiss and Polosa, though, as mentioned in the first section of the Results, they did not need a hypovolemic stimulus. We also have independent evidence for a central origin of the Mayer waves here, in that the motor actions were fictive and, in the majority of cases where the Mayer waves appeared as isolated increases from a flat baseline, the first event was an increase in the phrenic discharge, before the rise in blood pressure occurred, similar to the phase difference reported by Preiss et al. (1975), also for fictive respiration in decerebrate preparations.

A different question is related to the identity of the descending pathways mediating the activation of the preganglionic sympathetic neurons. It has been demonstrated that neurons of the caudal raphe nuclei project to the intermediolateral cell column (Bacon et al., 1990), but investigations on the serotonergic actions on the sympathetic activity has been dogged by methodological problems (cf. review by Lovick, 1997), although there is evidence for a 5-HT-2-mediated excitation (Orer et al., 1996). There is also evidence for Mayer-wave related modulation in several of the caudal raphe neurons. Bruinstroop et al. (2012) have more recently documented a strong projection from the locus coeruleus onto the thoracic intermediolateral cell column where most of the cells are excited by α<sup>1</sup> receptors (Lewis and Coote, 1990a,b; see also Samuels and Szabadi, 2008). There is, however, no direct evidence for locus coeruleus neurons to be activated in relation to the Mayer waves.

Even though Mayer waves are correlated to efferent sympathetic activity there is so far no direct evidence for an increased descending monoaminergic drive to the spinal cord during these waves. It would be plausible that the brainstem activity during the Mayer waves is activating the preganglionic neurons in the intermediolateral cell column via descending glutamatergic pathways, which may also activate the spinal locomotor circuits. It is wellestablished that monoaminergic innervation of the spinal cord affect the intrinsic properties of the motoneurons, enhancing their PICs and thus their plateau properties (see e.g., Hounsgaard et al., 1988; Hounsgaard and Kiehn, 1989 for original articles and several subsequent reviews: Powers and Binder, 2001; Heckman et al., 2003, 2005, 2009; Hultborn et al., 2004; ElBasiouny et al., 2010; Zhang et al., 2012). However, the facilitation of the "plateau properties" in the motoneurons could possibly also be explained by activation of glutamatergic NMDA receptors (cf. Delgado-Lezama et al., 1997; Guertin and Hounsgaard, 1998; Enríquez Denton et al., 2012). It thus now seems warranted to directly investigate the monoaminergic levels during Mayer waves e.g., with voltametric techniques (cf. Hentall et al., 2006).

Further investigation is also warranted as to whether the behavior reported here represents a physiological mechanism or an epiphenomenon. The new observation here of the modulation of CPG interaction, and probably also of the motoneuron intrinsic properties, by a fast-acting descending, naturally occurring drive, is certainly a mechanism which could be physiologically useful. This drive is related, via the Mayer-waves to the control of blood pressure. In broad terms, control of respiration and blood pressure are regarded by Holstege as part of the "emotional motor system," which he describes as that involved in survival behaviors (Holstege, 1991). In this context, an association with limb motor control should be of no surprise. One particular aspect of that, related to plateau potentials evoked by the respiratory drive, has already been suggested (Kirkwood and Ford, 2004), but there could be many others. In the decerebrate preparation without any specific stimulation, only some features of that association are likely to be detectable.

## **AUTHOR CONTRIBUTIONS**

Conception and design of study: Jacob Wienecke, Manuel Enríquez Denton, Peter A. Kirkwood, Hans Hultborn. Acquisition, analysis and/or interpretation of data: Jacob Wienecke, Manuel Enríquez Denton, Katinka Stecina, Peter A. Kirkwood, Hans Hultborn. Drafting the ms.: Katinka Stecina, Peter A. Kirkwood, Hans Hultborn. Critical review of the ms. and its final approval: Jacob Wienecke, Manuel Enríquez Denton, Katinka Stecina, Peter A. Kirkwood, Hans Hultborn.

## **ACKNOWLEDGMENTS**

We thank Lillian Grøndahl, Allan Djørup and Egil Gudbrandsen for expert, long-provided technical assistance in our Copenhagen laboratory, and Gilles Detillieux, Winnipeg, Canada, for expert help with the data collection/analysis system. The research has been supported by grants from the Danish Medical Research Council, the Royal Society, the Ludvig and Sara Elsass's Foundation, the Sofus & Olga Friis Foundation and the International Spinal Cord Research Trust. K. S. was supported by EU FP6—Marie Curie Fellowship.

## **REFERENCES**

stimulation of the nucleus raphe magnus. *Neuroscience* 142, 893–903. doi: 10.1016/j.neuroscience.2006.06.038

Zhang, M., Hultborn, H., and Sukiasyan, N. (2012). "The distribution of calcium channel Cav1.3 in the central nervous system and its functions in relation to motor control," in *Calcium Channels: Properties, Functions and Regulation*, ed M. R. Figgins (Hauppauge, NY: Nova Science Publishers), 1–48.

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

*Received: 21 July 2014; accepted: 05 January 2015; published online: 10 February 2015.*

*Citation: Wienecke J, Enríquez Denton M, Stecina K, Kirkwood PA and Hultborn H (2015) Modulation of spontaneous locomotor and respiratory drives to hindlimb motoneurons temporally related to sympathetic drives as revealed by Mayer waves. Front. Neural Circuits 9:1. doi: 10.3389/fncir.2015.00001*

*This article was submitted to the journal Frontiers in Neural Circuits.*

*Copyright © 2015 Wienecke, Enríquez Denton, Stecina, Kirkwood and Hultborn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

REVIEW ARTICLE published: 11 February 2015 doi: 10.3389/fncir.2015.00002

# Spinal metaplasticity in respiratory motor control

## **Daryl P. Fields and Gordon S. Mitchell \***

Department of Comparative Biosciences, University of Wisconsin-Madison, Madison, WI, USA

#### **Edited by:**

Hans Hultborn, University of Copenhagen, Denmark

#### **Reviewed by:**

Deborah Baro, Georgia State University, USA David Parker, Cambridge University, UK Carlos Mantilla, Mayo Clinic, USA

#### **\*Correspondence:**

Gordon S. Mitchell, Department of Comparative Biosciences, University of Wisconsin-Madison, 2015 Linden Drive, Madison, WI 53706, USA e-mail: mitchell@ svm.vetmed.wisc.edu

A hallmark feature of the neural system controlling breathing is its ability to exhibit plasticity. Less appreciated is the ability to exhibit metaplasticity, a change in the capacity to express plasticity (i.e., "plastic plasticity"). Recent advances in our understanding of cellular mechanisms giving rise to respiratory motor plasticity lay the groundwork for (ongoing) investigations of metaplasticity. This detailed understanding of respiratory metaplasticity will be essential as we harness metaplasticity to restore breathing capacity in clinical disorders that compromise breathing, such as cervical spinal injury, motor neuron disease and other neuromuscular diseases. In this brief review, we discuss key examples of metaplasticity in respiratory motor control, and our current understanding of mechanisms giving rise to spinal plasticity and metaplasticity in phrenic motor output; particularly after pre-conditioning with intermittent hypoxia. Progress in this area has led to the realization that similar mechanisms are operative in other spinal motor networks, including those governing limb movement. Further, these mechanisms can be harnessed to restore respiratory and non-respiratory motor function after spinal injury.

**Keywords: respiratory control, respiratory plasticity, metaplasticity, spinal cord, motor neuron, intermittent hypoxia, phrenic motor neuron**

## **INTRODUCTION**

As with most neural systems, a hallmark of the neural system controlling breathing is its ability to express plasticity; defined as a persistent (>60 min) change in neural network function after an experience/stimulus has ended (Mitchell and Johnson, 2003). Respiratory plasticity is characteristic of development, continues throughout life, and is of considerable importance in preserving life when confronted with clinical disorders that compromise the ability to breathe, including diseases of the lung and chest wall, or neurological disorders such as spinal injury and motor neuron disease (Vinit et al., 2009; Dale-Nagle et al., 2010; Nichols et al., 2013; Dale et al., 2014).

Less well known is that respiratory plasticity itself adapts based on experience (Mitchell and Johnson, 2003), a phenomenon referred to as metaplasticity or "plastic plasticity" (Abraham and Bear, 1996). Although there are examples of respiratory plasticity and metaplasticity in sensory receptors, including the carotid body (CB) chemoreceptors (Kumar and Prabhakar, 2012); prominent examples of respiratory plasticity and metaplasticity are found at the other end of the respiratory control system, the motor nuclei (Mahamed and Mitchell, 2007; Dale-Nagle et al., 2010; Devinney et al., 2013).

In this brief review, we focus on known examples of spinal respiratory motor plasticity and metaplasticity, as well as implications for clinical application. We first define respiratory plasticity, metaplasticity and related concepts, and then the potential sites of respiratory plasticity, models of spinal respiratory plasticity/metaplasticity, possible mechanisms of metaplasticity, and finally, gaps in our knowledge that require additional research.

## **DEFINITIONS: (META) PLASTICITY AND (META) MODULATION**

Modulation and plasticity are related but distinct properties that are frequently confused. Higher order properties such as metamodulation and metaplasticity are less frequently considered and even more often confused. Thus, brief definitions of these terms are provided in the context of respiratory motor control (Mitchell and Johnson, 2003); although they also apply to limb motor control (reviewed in Grau et al., 2014).

*Modulation* is a change in system behavior that fades rapidly (seconds to minutes) after the stimulus is removed. Neuromodulators often work through metabotropic G protein coupled receptors, which alter cell excitability through covalent modifications of membrane channels. Modulation does confer system flexibility and can initiate cellular mechanisms resulting in plasticity (Mitchell and Johnson, 2003), but the two are differentiated by what happens when the stimulating trigger is removed. For example, during a brief (5 to 30 min) hypoxic experience, phrenic nerve activity (and breathing) increases and returns to normal seconds/minutes after hypoxia has ceased. Alternatively, 3 successive 5-min hypoxic episodes give rise to a persistent increase in phrenic nerve activity lasting several hours after the final hypoxic episode has ended—an expression of plasticity (see below). In this example, modulation is the within episode (during hypoxia) augmentation of respiratory motor output, plasticity is the persistence of increased activity that lasts long after the hypoxia stimulus has ended.

*Meta-modulation* is a reversible change in the capacity or quality of modulation (Katz and Edwards, 1999; Mitchell and Johnson, 2003), and requires continued presence of the meta-modulation trigger. Meta-modulating stimuli also frequently act through G protein coupled receptors, 2nd messengers and/or ion channels to augment the response of neurons to modulators (Katz and Edwards, 1999; Mesce, 2002; Ribeiro and Sebastiao, 2010), though metamodulation triggers have not yet been associated with long-term changes in gene expression. One interesting example of meta-modulation in respiratory control is the response of neurons in the nucleus of the solitary tract to concurrent serotonin and substance P application (Jacquin et al., 1989). Both serotonin and substance P alone positively modulate nucleus tractus solitarius (NTS) neurons. However, in the presence of substance P, serotonin becomes inhibitory (Jacquin et al., 1989). Thus, in these *in vitro* conditions, the impact of one modulator (serotonin) is augmented by concurrent application of another (substance P).

*Plasticity (long-term)* is a persistent (>60 min) change in function that outlasts the initiating stimulus (Mitchell and Johnson, 2003). Plasticity often requires new protein synthesis via translational and/or transcriptional regulation (Manahan-Vaughan et al., 2000; Mitchell and Johnson, 2003; Alberini, 2008), though it is not a prerequisite. A frequent initiating stimulus in many neural systems is neuronal activity, or activity-dependent synaptic plasticity (Malinow and Malenka, 2002; Wiegert and Bading, 2011); though activity-dependent plasticity is not characteristic of respiratory motor control (Mitchell and Johnson, 2003; Strey et al., 2013). Instead, neuromodulators frequently elicit respiratory plasticity through distinct signaling cascades induced by patterned metabotropic receptor activation.

One prominent model of plasticity in spinal respiratory motor control is phrenic long-term facilitation (pLTF), a long-lasting increase in phrenic motor output observed following acute intermittent hypoxia (AIH; Feldman et al., 2003; Mahamed and Mitchell, 2007; Dale-Nagle et al., 2010; Devinney et al., 2013). AIH elicits episodic serotonin release within the phrenic motor nucleus (Kinkead et al., 2001), activation of spinal serotonin receptors (Fuller et al., 2001; Baker-Herman and Mitchell, 2002) and a long-lasting enhancement of phrenic motor output (Mahamed and Mitchell, 2007; Devinney et al., 2013). Since this form of phrenic motor plasticity is initiated by intermittent, but not sustained hypoxia of similar cumulative duration, it is pattern sensitive (Baker and Mitchell, 2000; Devinney et al., 2013), similar to other forms of serotonin dependent plasticity (Sherff and Carew, 2002; Philips et al., 2013). In summary, modulatory experiences in themselves are not sufficient for plasticity as their effects fade once the trigger is removed. Alternatively, pattern specific modulation can be encoded through discrete signaling pathways to elicit a long-lasting augmentation of nerve output that persists after the triggering experience; i.e., plasticity/metaplasticity.

*Metaplasticity* is a change in the capacity for neuroplasticity (Abraham and Bear, 1996; Byrne, 1997). An important distinction between metaplasticity and metamodulation is that metaplasticity is expressed after triggering experiences (i.e., hypoxia) for both plasticity and metaplasticity are gone (Abraham, 2008). As in plasticity, metaplasticity often encodes previous experiences by altering gene expression (Mitchell and Johnson, 2003); therefore changing the ability of a system to respond to subsequent experiences. Through encoded memory, the effects of metaplasticity triggers can persists long after the stimulation has subsided; the focus of this review is to review how intermittent hypoxia training (a metaplasticity trigger) can augment subsequent spinal respiratory plasticity.

## **POTENTIAL SITES OF RESPIRATORY PLASTICITY AND METAPLASTICITY**

Essential processes in control of breathing include respiratory rhythm generation, burst pattern formation (Feldman and Smith, 1989; Mitchell et al., 1990) as well as sensory feedback giving rise to chemoreflexes, mechanoreflexes, neuromodulation and neuroplasticity (Feldman et al., 2003; Mitchell and Johnson, 2003).

Respiratory plasticity and metaplasticity can occur in any component of the neural system controlling breathing (Mitchell and Johnson, 2003). Of particular interest to this review, plasticity and metaplasticity occur in spinal respiratory motor neurons (and/or interneurons) putting the final "touches" on burst pattern formation before the central nervous system (CNS) relays its command to breathe to respiratory muscles. Thus, plasticity and metaplasticity are important in sculpting motor output to individual respiratory muscles. Here, we focus on spinal respiratory motor plasticity, with less consideration given to plasticity in chemoreceptor feedback (Bisgard, 2000; Kumar and Prabhakar, 2012) or brainstem mechanisms of respiratory rhythm generation/pattern formation (Blitz and Ramirez, 2002; Feldman et al., 2003, 2005). We instead refer to several well-written reviews of carotid (Kumar and Prabhakar, 2012) and brainstem (Ramirez et al., 2012) plasticity, which can elicit upstream respiratory plasticity that is phenotypically similar to spinal respiratory plasticity.

One prominent source of modulatory input giving rise to plasticity originates from brainstem serotonergic raphe neurons that project to spinal and brainstem respiratory neurons. In particular, brainstem raphe neurons are known to modulate respiratory motor neurons and, under the right circumstances, initiate important forms of respiratory motor plasticity (Mitchell et al., 2001; Feldman et al., 2003). **Figure 1** outlines the neural network controlling breathing and identifies potential sites of respiratory neuroplasticity; all of which could also exhibit metaplasticity.

## **PHRENIC LONG-TERM FACILITATION IS A FORM OF SPINAL RESPIRATORY MOTOR PLASTICITY**

The most extensively studied model of spinal respiratory motor plasticity is AIH induced pLTF; a persistent increase in phrenic nerve output following 3 × 5 min experiences of moderate hypoxia (35–45 mmHg PaO2; Hayashi et al., 1993; Bach and Mitchell, 1996; Mitchell et al., 2001; Feldman et al., 2003; Mahamed and Mitchell, 2007; Dale-Nagle et al., 2010; Devinney et al., 2013). In recent years we have come to use a generic term for long-lasting enhancement of phrenic motor output; phrenic motor facilitation (pMF). Whereas pMF may arise from a variety of triggers (i.e., inactivity, hypercapnia, pharmacology), pLTF

is a specific form of pMF elicited from AIH (Devinney et al., 2013).

In many instances, spinal signaling pathways that are necessary for pLTF are also independently sufficient to elicit pMF (reviewed by Dale-Nagle et al., 2010). For example, serotonin receptor antagonists applied to the C3–C5 cervical spine region during AIH abolishes pLTF; demonstrating that spinal serotonin receptor activation in the immediate vicinity of the phrenic motor nucleus is necessary for pLTF (Fuller et al., 2001; Baker-Herman and Mitchell, 2002). AIH-induced pLTF is also abolished by cervical spinal pre-treatment with siRNAs targeting BDNF mRNA (Baker-Herman et al., 2004) and neuronal nitric oxide synthase (nNOS; MacFarlane et al., 2014). Conversely, activation of cervical spinal serotonin type 2A, 2B and 7 receptors (MacFarlane and Mitchell, 2009; Hoffman and Mitchell, 2011; MacFarlane et al., 2011), TrkB receptors (the high affinity brain derived neurotrophic factor (BDNF) receptor; Baker-Herman et al., 2004), and nNOS (MacFarlane et al., 2014) can all give rise to phenotypically similar pMF in the absence of hypoxia (i.e., mimicking pLTF). Collectively, these experiments reveal important aspects of the signaling pathway giving rise to AIH-induced pLTF, but also identify important signaling checkpoints that are independently sufficient for pMF.

Though similar spinal signaling pathways can drive pLTF and other forms of pMF, it is important to differentiate between the two experimentally and conceptually (Dale-Nagle et al., 2010). For example, intrathecal drug application allows for the investigation of pMF mechanisms confined to the region of the phrenic motor nucleus. Conversely, moderate AIHinduced pLTF has global effects that influence other respiratory control sites (**Figure 1**) as well as non-respiratory motor pools within the thoracic and lumbar spinal segments. AIH-induced pLTF begins via hypoxia activation of peripheral (caotid body) chemoreceptors (Hayashi et al., 1993; Bach and Mitchell, 1996; Bavis and Mitchell, 2003), second order medullary neurons in the nucleus of the solitary tract, and then (indirectly) serotonergic neurons of the medullary raphe nuclei (Morris et al., 1996; Li et al., 1999; Mitchell et al., 2001; **Figure 1**). Subsequent episodic serotonin release on or near spinal respiratory motor neurons (Kinkead et al., 2001) initiates cellular cascades giving rise to moderate AIH (35–45 mmHg PaO2) induced pLTF (Fuller et al., 2001).

When elicited by more severe hypoxic episodes (25–30 mmHg PaO2) but otherwise the same AIH protocol (Nichols et al., 2012), AIH elicits pLTF through an entirely different mechanism; requiring spinal adenosine type 2A (A2A) receptor activation (Golder et al., 2008; Nichols et al., 2012). Our working model is that severe hypoxia triggers greater adenosine triphosphate (ATP) release from CNS cells, increasing ATP and adenosine concentrations. Elevated adenosine near phrenic motor neurons (PMN) activates A2A receptors and drives adenosine-dependent (serotonin independent) pLTF (Nichols et al., 2012). We have not yet identified the specific cellular source of adenosine for severe AIH induced pLTF. However, astrocytes increase their release of ATP and adenosine during hypoxia, and are a likely source for the relevant adenosine for severe AIH-induced pLTF (Kulik et al., 2010). Additional research is needed to confirm this hypothesis.

Although serotonin- and adenosine-dependent pLTF are phenotypically similar, they operate through distinct signaling pathways referred respectively as the Q and S pathways to pMF (Dale-Nagle et al., 2010); these pathways are named for the G proteins most often coupled to their initiating receptors (i.e., Gq for serotonin type 2 and Gs for adenosine 2A). Additionally, pMF can be induced by activation of Gq-coupled alpha-1 adrenergic receptors (Huxtable et al., 2014) or Gs-coupled 5-HT7 receptors (Hoffman and Mitchell, 2013). However, neither alpha-1 or 5-HT7 receptors are necessary for any form of AIH-induced pLTF (Hoffman and Mitchell, 2013; Huxtable et al., 2014). Thus, these receptors are sufficient for pMF, but not necessary for pLTF; they elicit pMF by convergent downstream signaling onto Q and S pathways.

The Q pathway to pMF consists of: spinal Gq-linked G protein coupled receptors activation (Fuller et al., 2001; Baker-Herman and Mitchell, 2002; MacFarlane et al., 2011; Huxtable et al., 2014), protein kinase C (PKC) activation (Devinney and Mitchell, unpublished observations), new synthesis of BDNF (Baker-Herman and Mitchell, 2002; Baker-Herman et al., 2004), TrkB receptors (Baker-Herman et al., 2004; Dale et al., unpublished), and downstream signaling via ERK MAP kinases (Wilkerson and Mitchell, 2009; Hoffman et al., 2012). In contrast, the S pathway to pMF involves: spinal Gs-linked G protein coupled receptors (Hoffman and Mitchell, 2011; Nichols et al., 2012), adenylyl cyclase activation with synthesis of cyclic AMP, new protein synthesis of an immature TrkB isoform (vs. BDNF; Golder et al., 2008; Hoffman and Mitchell, 2011) and downstream signaling via Akt (vs. ERK; Golder et al., 2008; Hoffman et al., 2012).

The Q and S pathways interact in interesting and complex ways that we have defined as "cross-talk inhibition" (Dale-Nagle et al., 2010; Hoffman et al., 2010; Hoffman and Mitchell, 2013). Our working hypothesis is that manipulations of these cross-talk interactions during intermittent hypoxia training may underlie at least some forms of spinal respiratory metaplasticity (see below). During moderate AIH-induced pLTF, serotonin release activates abundant, high affinity 5-HT2 receptors; driving the dominant Q pathway to pLTF. Although concurrent activation of Gs protein linked A2A or 5-HT7 receptors is insufficient to trigger the S pathway at these levels of hypoxia (moderate AIH-induced pLTF is exclusively Q pathway dependent plasticity), sub-threshold activation of these Gs-linked receptors constrains the Q pathway and reduces pLTF magnitude. Inhibition of spinal A2A receptors (Hoffman et al., 2010) and/or 5-HT7 receptors (Hoffman and Mitchell, 2013) during moderate AIH eliminates this cross-talk constraint thereby enhancing moderate AIH-induced pLTF. We suspect that all Gs-linked G protein coupled receptors have a capacity to constrain the Q pathway to pMF since cross-talk inhibition is mediated by protein kinase A (PKA); a prominent effector of downstream Gs protein/cAMP signaling. Whereas spinal PKA inhibition relieves cross-talk inhibition and enhances moderate AIH induced pLTF (to a similar extent as A2A and 5-HT7 inhibition), PKA activation suppresses moderate AIHinduced pLTF (Hoffman and Mitchell, 2013). Since Gs-linked G protein coupled receptors are commonly expressed within many CNS cell types, we cannot yet state that cross-talk inhibition is operative within a single neuron; spinal motor neurons. It remains possible that elements of the Q and S pathways, and their interactions, arise from interneuron, astrocyte and/or microglia. The cellular localization of processes involved in spinal respiratory plasticity is an area that requires and warrants additional research.

Independent from the contributions of G protein coupled receptors, spinal activation of JAK2-coupled erythropoietin (EPO; Dale et al., 2012) or tyrosine kinase coupled vascular endothelial growth factor (VEGF; Dale-Nagle et al., 2011) receptors is sufficient to elicit pMF. EPO, VEGF, and their high affinity receptors are all oxygen-regulated proteins with increased expression following prolonged hypoxia (Semenza et al., 1991; Forsythe et al., 1996). We suspect that these proteins play a relatively greater role in phrenic motor plasticity during conditions of prolonged hypoxia experiences (days-months). Both VEGF- and EPO-induced pMF are Akt and ERK dependent, suggesting downstream divergence onto both the Q and S signaling pathways (Dale-Nagle et al., 2011; Dale et al., 2012). The diversity of hypoxia-induced mechanisms giving rise to phenotypically similar pMF most likely reflects the need to adapt to hypoxia that differs in quality (severity, pattern) and duration. Specifically, we suggest that differential responses with severity (i.e., Q to S pathways) and duration (serotonin/adenosine to VEGF/EPO; Dale et al., 2014) enable unique interactions for adaptive flexibility during hypoxia. Further, this diversity of responses may enable emergent plasticity properties, such as metaplasticity.

# **pLTF METAPLASTICITY INDUCED BY INTERMITTENT HYPOXIA PRECONDITIONING**

## **CHRONIC INTERMITTENT HYPOXIA (CIH)**

CIH in rodents simulates some aspects of the intermittent hypoxia experienced during obstructive sleep apnea (OSA) in humans (Lee et al., 2009). Thus, when rodents are preconditioned with CIH, pathogenesis ensues (Reeves and Gozal, 2006; Moraes et al., 2012; Prabhakar and Semenza, 2012; Navarette-Opazo and Mitchell, 2014), yet multiple distinct forms of respiratory plasticity can also be observed. These include: (1) increased baseline breathing (Ling et al., 2001); (2) increased baseline carotid sinus chemoafferent neuron activity (Peng and Prabhakar, 2003, 2004); (3) decreased synaptic strength from carotid chemoafferent neurons onto their NTS targets (Kline et al., 2007); and (4) increased short-term hypoxic phrenic response (Ling et al., 2001; Peng and Prabhakar, 2003, 2004). In addition, CIH also triggers metaplasticity, expressed as enhanced AIH-induced pLTF (Ling et al., 2001), increased tidal volume in response to AIH within unanesthetized rats (i.e., ventilatory LTF; McGuire et al., 2003, 2004), and sensory LTF of carotid chemoafferents neurons (sensory LTF); a form of plasticity observed only after CIH preconditioning (**Figure 2**; Peng and Prabhakar, 2003, 2004). In normal rodents, AIH does not elicit sensory LTF; after CIH pretreatment, robust sensory LTF is observed after the same AIH stimulus (i.e., persistent enhancement of carotid chemoafferent activity; Peng and Prabhakar, 2003, 2004). Although the newly revealed sensory LTF may contribute to the CIH-enhanced AIHinduced pLTF, considerable evidence suggests that this is not the entire story.

Enhanced pLTF could occur and/or be modified at any site in the neural network transmitting chemosensory activity to PMN; including chemoreceptors themselves, their afferent terminations in the nucleus of the solitary tract, neurons of the ventral respiratory column, raphe neurons, spinal phrenic interneurons and/or PMN (**Figure 1**). There is currently no published evidence that CIH-induced pLTF metaplasticity results from changes in brainstem modulatory neurons, although prolonged CIH (four, but not one week; Bach and Mitchell, unpublished observations) increases serotonergic terminal density in the phrenic motor nucleus; suggesting that anatomical plasticity of medullary raphe neuron projections may contribute to enhanced pLTF (McCrimmon et al., 1995; Kinkead et al., 1998). Further evidence for CIH-induced CNS plasticity (vs. at the peripheral chemoreceptors) and pLTF metaplasticity includes observations that CIH: (1) increases phrenic nerve responses to electrical activation of the cut, central end of the carotid sinus nerve, bypassing carotid chemoreceptors *per se* (Ling et al., 2001); (2) attenuates synaptic input into the nucleus of the solitary tract (i.e., wrong way to account for enahnced pLTF; Kline et al., 2007,

2009); and (3) strengthens spinal pathways to PMN following cervical injury (Fuller et al., 2003).

Although relatively little is known concerning sites and signaling mechanisms of CIH metaplasticity, CIH-enhanced moderate AIH-induced pLTF is still serotonin-dependent (Ling et al., 2001; McGuire et al., 2004). On the other hand, it is no longer dependent on 5-HT2 receptors alone (Ling et al., 2001; McGuire et al., 2004). Rather, the inhibitory actions of 5-HT7 receptors on moderate AIH-induced pLTF seen in normal rats (Hoffman and Mitchell, 2013) convert to excitatory actions; both 5-HT7 and 5-HT2 receptors now contribute to enhanced LTF in CIH trained rats (McGuire et al., 2004). Thus, CIH may disrupt normal cross-talk interactions between the Q and S pathways without compromising the respective Q and S contributions to pMF (enabling additive contributions). Mechanisms accounting for this apparent loss of cross-talk inhibition between 5-HT2 and 5-HT7 receptors are not known.

Because CIH elicits respiratory metaplasticity, we initially had contemplated CIH as a treatment option to restore respiratory control in clinical disorders that impair breathing capacity; such as cervical spinal injury (Fuller et al., 2003; Mitchell, 2007). Unfortunately, CIH has well-documented adverse consequences in multiple physiological systems; including systemic hypertension (Fletcher et al., 1992), metabolic syndrome (Li et al., 2005; Tasali and Ip, 2008) and hippocampal cell death with associated cognitive deficits (Row, 2007; Row et al., 2007). Thus, although CIH may have beneficial effects in restoring respiratory control in clinical disorders that compromise breathing, undesirable side effects limit its utility. An interesting corollary is that the increased sleep apnea prevalence in individuals with chronic spinal injuries (Sankari et al., 2014) may represent a form of "self medication," inducing spontaneous functional recovery. However, other factors associated with sleep apnea such as sleep fragmentation and systemic inflammation may over-ride any benefits (McGuire et al., 2008; Hakim et al., 2012; Zhang et al., 2012; Huxtable et al., 2013).

## **REPETITIVE ACUTE INTERMITTENT HYPOXIA (rAIH)**

Less intensive rAIH preconditioning protocols retain the ability to elicit plasticity and metaplasticity without the known pathogenic effects of CIH (Lovett-Barr et al., 2012; Satriotomo et al., 2012; Navarette-Opazo and Mitchell, 2014). For example, daily acute intermittent hypoxia (dAIH; 10, 5 min hypoxic episodes for seven consecutive days) increases the strength of crossedspinal synaptic pathways to PMN (Lovett-Barr et al., 2012) and enhances moderate AIH-induced LTF (Wilkerson and Mitchell, 2009). Similarly, AIH three times per week (3xwAIH; 10, 5 min hypoxic episodes, three times per week for four or 10 weeks) increases the expression of pro-plasticity molecules within PMN (Satriotomo et al., 2012), and enhances moderate AIH-induced pLTF (MacFarlane et al., 2010; Vinit et al., 2010). In contrast to CIH, neither dAIH (Wilkerson and Mitchell, 2009; Lovett-Barr et al., 2012) or 3xwAIH (Satriotomo et al., 2012) elicit detectable systemic hypertension, hippocampal cell death or reactive gliosis. With similar "low dose" intermittent hypoxia protocols, most reported systemic effects are in fact beneficial vs. pathogenic (Dale et al., 2014; Navarette-Opazo and Mitchell, 2014). Thus, rAIH may be a simple, safe and effective means of promoting motor plasticity and functional recovery of breathing (and nonrespiratory motor) deficits caused by severe clinical disorders. For example, dAIH beginning one week after cervical spinal hemisection in rats promotes remarkable functional recovery of breathing capacity (Lovett-Barr et al., 2012). Even more startling, dAIH in combination with ladder walking induced prolonged restoration of forelimb function in rats (Lovett-Barr et al., 2012). The functional benefits of intermittent hypoxia therapy may be most notable when paired with task specific training. For example, dAIH for five days increased walking endurance by 18% in humans with chronic (>nine months), incomplete spinal injuries, but increased walking endurance 38% when combined with walking practice. Conversely, walking practice alone did not improve walking endurance (Hayes et al., 2014). Although detailed mechanisms underlying rAIH induced respiratory and limb functional recovery after spinal injury are not yet known, increased expression of molecules known to be associated with both the Q and S pathways suggests that both signaling pathways may be involved (Wilkerson and Mitchell, 2009; Lovett-Barr et al., 2012; Satriotomo et al., 2012; Dale et al., 2014). Furthermore, with transcriptome analysis of gene arrays from ventral cervical segments encompassing the phrenic motor nucleus, the most significant changes noted were associated with anti-inflammatory activities. Specifically, there was a pronounced reduction in activity of the pro-inflammatory transcription factors NfκB and Stat 1/2 (Small et al., 2014).There is little direct evidence to date concerning mechanisms of rAIH-induced pLTF metaplasticity; but these studies have provided several promising leads. Additional mechanistic studies are critical to understand

pLTF is doubled in normal rats when cross-talk inhibition from the S pathway is reduced from cervical spinal inhibition of A2A receptors, 5-HT7 receptors or PKA (see text for references). In either case, the Q pathway remains the

this highly novel and clinically translatable phenomenon. **Figure 3** outlines several possible changes in inter-pathway interactions that may enhance pLTF following rAIH training. A greater understanding of rAIH-induced metaplasticity will be important as we develop rAIH into a therapeutic tool to treat motor deficits caused by spinal injury (Lovett-Barr et al., 2012; Trumbower et al., 2012; Hayes et al., 2014) or motor neuron disease (Mitchell, 2007; Nichols et al., 2013).

## **OTHER MODELS OF pLTF METAPLASTICITY?**

AIH-induced pLTF is modified by a number of other pretreatments that enhance and diminish its expression (Mitchell et al., 2001). However, since it is not always possible to reverse the initiating factor/treatment (i.e., metaplasticity trigger), it is sometimes ambiguous if the change in pLTF reflects true metaplasticity vs. a persistent change in the initiating stimulus. Conditions altering AIH-induced pLTF that we suspect reflect models of metaplasticity include: (1) chronic cervical supported by available evidence concerning mechanisms of enhanced pLTF after CIH pre-conditioning, where both 5-HT2 and 5-HT7 receptors appear to make independent contributions (see text).

dorsal rhizotomy (CDR; Kinkead et al., 1998); (2) systemic inflammation (Vinit et al., 2011; Huxtable et al., 2013); (3) cervical spinal injury in a time-dependent manner (Golder and Mitchell, 2005); (4) ALS (Nichols and Mitchell, 2014); (5) age (Zabka et al., 2001); and (6) sex hormones (Zabka et al., 2006).

Of these, the best characterized is enhanced pLTF following CDR. Twenty-eight days following bilateral CDR, moderate AIH induced pLTF is doubled, with an equivalent increase in serotonin terminal density in the immediate vicinity of identified PMN (Kinkead et al., 1998). Increased serotonin terminal density is associated with enhanced pLTF in several models of pLTF metaplasticity (see above, McCrimmon et al., 1995; Kinkead et al., 1998; Mitchell et al., 2001; Satriotomo et al., 2006). Both normal and CDR-enhanced pLTF are abolished by the 5-HT2 receptor antagonist, ketanserin, demonstrating that CDR-enhanced pLTF arises from accentuation of normal Q pathway dependent pLTF (vs. combined Q/S pathway contributions following CIH training).

One week post-CDR, ventral spinal BDNF and neurotrophin-3 concentrations are increased (Johnson et al., 2000), as is the strength of crossed spinal synaptic pathways to PMN (Fuller et al., 2002). Similarly, end-stage motor neuron disease (ALS) is associated with: (1) increased BDNF and other growth/trophic factor expression in PMN (Satriotomo et al., 2006); (2) increased serotonin terminal density; and (3) enhanced Q pathway dependent pLTF (Nichols and Mitchell, 2014). Thus, enhanced pLTF may occur via accentuation of the normal Q pathway to pLTF (i.e., following CDR and ALS), or additive contributions from the Q and S pathways to pMF (e.g., following CIH). Several proposed mechanisms for enhanced, AIH-induced pLTF are outlined within **Figure 3**.

## **SIGNIFICANCE**

Although considerable effort has been devoted to investigations of detailed cellular mechanisms giving rise to AIH-induced pLTF (Devinney et al., 2013), relatively little attention has been given to the equally important concept of respiratory metaplasticity. We still lack a fundamental understanding of when, where and how respiratory metaplasticity occurs. Even though metaplasticity confers a remarkable potential to amplify (and harness) existing mechanisms of plasticity, metaplasticity also offers promise to reveal new forms of plasticity not present in normal animals. Examples of the latter include sensory LTF after CIH preconditioning (Peng and Prabhakar, 2003, 2004), and dAIH-revealed, AIH-induced hypoglossal LTF in Brown Norway rats; a strain that does not normally exhibit hypoglossal LTF (Wilkerson and Mitchell, 2009). While hypoglossal LTF and pLTF are phenotypically similar (i.e., enhanced nerve activity following prior experience), the threshold for eliciting each differs based on triggering stimulus and strain/species (reviewed in Golder et al., 2005), strongly suggesting an adaptive change in plasticity capacity/threshold following dAIH training.

With our significant progress in understanding cellular mechanisms of AIH-induced pLTF, it is an advantageous model to study metaplasticity in respiratory motor control. Such studies are warranted from a basic science perspective, but also because rAIH is rapidly moving towards clinical application as a treatment for respiratory insufficiency in disorders that compromise breathing capacity (Lovett-Barr et al., 2012; Nichols et al., 2013) as well as motor deficits in non-respiratory motor systems (Lovett-Barr et al., 2012; Hayes et al., 2014). Principles elucidated by studies of plasticity and metaplasticity in respiratory motor control may be an essential guide for understanding the plasticity, metaplasticity and functional recovery of a diverse range of clinical disorders that compromise movement (Lovett-Barr et al., 2012; Dale et al., 2014; Hayes et al., 2014).

## **REFERENCES**

plasticity in the phrenic motor nucleus. *Exp. Neurol.* 237, 103–115. doi: 10. 1016/j.expneurol.2012.05.020

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

*Received: 21 July 2014; accepted: 07 January 2015; published online: 11 February 2015*. *Citation: Fields DP and Mitchell GS (2015) Spinal metaplasticity in respiratory motor control. Front. Neural Circuits 9:2. doi: 10.3389/fncir.2015.00002*

*This article was submitted to the journal Frontiers in Neural Circuits*.

*Copyright © 2015 Fields and Mitchell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution and reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms*.

# Sensory Activation of Command Cells for Locomotion and Modulatory Mechanisms: Lessons from Lampreys

Gheylen Daghfous 1,2 , Warren W. Green3† , Simon T. Alford<sup>4</sup> , Barbara S. Zielinski <sup>3</sup> and Réjean Dubuc1,2 \*

<sup>1</sup> Groupe de Recherche en Activité Physique Adaptée, Département des Sciences de l'Activité Physique, Université du Québec à Montréal, Montréal, QC, Canada, <sup>2</sup> Groupe de Recherche sur le Système Nerveux Central, Département de Neurosciences, Université de Montréal, Montréal, QC, Canada, <sup>3</sup> Department of Biological Sciences and Great Lakes Institute for Environmental Research, University of Windsor, Windsor, ON, Canada, <sup>4</sup> Laboratory of Integrative Neuroscience, Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL, USA

Sensorimotor transformation is one of the most fundamental and ubiquitous functions of the central nervous system (CNS). Although the general organization of the locomotor neural circuitry is relatively well understood, less is known about its activation by sensory inputs and its modulation. Utilizing the lamprey model, a detailed understanding of sensorimotor integration in vertebrates is emerging. In this article, we explore how the vertebrate CNS integrates sensory signals to generate motor behavior by examining the pathways and neural mechanisms involved in the transformation of cutaneous and olfactory inputs into motor output in the lamprey. We then review how 5-hydroxytryptamine (5-HT) acts on these systems by modulating both sensory inputs and motor output. A comprehensive review of this fundamental topic should provide a useful framework in the fields of motor control, sensorimotor integration and neuromodulation.

#### Edited by:

Brian R. Noga, University of Miami, USA

#### Reviewed by:

Joseph Fetcho, Cornell University, USA Larry M. Jordan, University of Manitoba, Canada

#### \*Correspondence:

Réjean Dubuc<EMAIL_ADDRESS>
## †Present address:

Warren W. Green, Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL, USA

Received: 25 November 2015 Accepted: 07 March 2016 Published: 22 March 2016

#### Citation:

Daghfous G, Green WW, Alford ST, Zielinski BS and Dubuc R (2016) Sensory Activation of Command Cells for Locomotion and Modulatory Mechanisms: Lessons from Lampreys. Front. Neural Circuits 10:18. doi: 10.3389/fncir.2016.00018 Keywords: sensorimotor, locomotion, modulation, reticulospinal neurons, lamprey, 5-HT

# INTRODUCTION

Locomotion is a rhythmic motor behavior involved in everyday functions. It requires the activation and coordination of the axial and/or appendicular musculature. Spinal neuronal networks called ''central pattern generators'' (CPGs) for locomotion generate the patterns of muscle activation that underlie propulsion during locomotion. Supraspinal structures, on the other hand, are required

**Abbreviations:** 5-HT, 5-hydroxytryptamine; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AOO, accessory olfactory organ; ARRN, anterior rhombencephalic reticular nucleus; CPG, central pattern generator; DR, dorsal root; dV, descending root of the trigeminal nerve; DCN, dorsal column nucleus; EPSC, excitatory post synaptic current; EPSP, excitatory post synaptic potential; MLR, mesencephalic locomotor region; MOE, main olfactory epithelium; MRN, mesencephalic reticular nucleus; MRRN, middle rhombencephalic reticular nucleus; ndV, nucleus of the descending root of the trigeminal nerve; NMDA, N-methyl-D-aspartate; NMDAR, N-methyl-D-aspartate receptor; OB, olfactory bulb; OLA, octavolateralis area; OSN, olfactory sensory neurons; PRRN, posterior rhombencephalic reticular nucleus; PT, posterior tuberculum; RS, reticulospinal; SC, spinal cord; SNAP-25, synaptosomal-associated protein 25; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TTX, tetrodotoxin; VGCC, voltage-gated calcium channel; VR, ventral root.

for activating and controlling the spinal CPGs. Descending inputs trigger, maintain, and eventually stop locomotion. The brainstem reticulospinal (RS) cells act as command cells that constitute an important interface between supraspinal and spinal networks. As such, the activation of RS cells by sensory (sensoryevoked locomotion) or internal clues (goal-directed locomotion) will markedly influence spinal function.

In this review, we focus on sensory-evoked locomotion by examining how two different sensory modalities influence the activation RS cells in a basal vertebrate, the lamprey. Because lampreys share a common brain ''bauplan'' with jawed vertebrates, including mammals, knowledge gained from neural circuits and mechanisms in lampreys provides insight into fundamental principles of vertebrate brain organization and function (Grillner et al., 1998b; Robertson et al., 2014). This review article focuses on some recent work in the lamprey from our labs on the pathways and neural mechanisms involved in the transformation of cutaneous and olfactory inputs into motor output. These sensory modalities are of paramount importance for the survival and reproductive success of individuals as they drive feeding, reproductive, and escape behaviors. We will also discuss 5-hydroxytryptamine (5-HT) modulation of these sensorimotor pathways. Indeed, 5-HT modulates both the sensory inputs to the RS cells at the supraspinal level and the descending motor commands of the RS cells in the spinal cord (SC). The mechanisms by which 5-HT modulates synaptic transmission has been well described in lampreys (Takahashi et al., 2001; Alford et al., 2003; Gerachshenko et al., 2005; Schwartz et al., 2005; Photowala et al., 2006; Schwartz et al., 2007; Gerachshenko et al., 2009; Alpert and Alford, 2013).

After a brief review of the motor circuitry and neural mechanisms of locomotion, sensorimotor transformations will be addressed starting with the neural pathways, from the receptors to the neural centers, followed by the neural mechanisms. Sensorimotor transformations of cutaneous and olfactory inputs will be addressed similarly. We have recently shown that the neural connections for these two sensory modalities differ considerably, yet activate the same target cells in the lower brainstem, the RS cells (Viana Di Prisco et al., 2000; Derjean et al., 2010). The pathway for cutaneous-induced locomotor reactions is shorter and more direct to the RS cells than the pathway involved in olfactory-induced locomotion. The transformation mechanisms are also different. The RS cell responses to cutaneous inputs (mechanical stimulation) switch from subthreshold excitatory postsynaptic potentials (EPSPs) to large sustained depolarizations; the output being an all-ornone escape locomotor bout. The olfactory inputs likely provide a more finely controlled locomotor output by acting via the mesencephalic locomotor region (MLR), a specific brainstem region involved in controlling goal directed locomotion. This review article will compare these two sensorimotor systems at the levels of neuronal connectivity and cellular mechanisms. We will then examine how 5-HT acts on these systems. In the past we have shown that cutaneous sensory inputs to RS cells are modulated by 5-HT (Antri et al., 2008). Similarly, there is prominent 5-HT innervation in the olfactory system, from the olfactory epithelium to the olfactory bulb (OB; Zielinski et al., 2000). Finally, we will review 5-HT modulation at the SC level. It has been extensively documented in the past that it exerts presynaptic effects that modulate the transmission from descending RS axons on SC neurons (Schwartz et al., 2005, 2007). This modulation has powerful effects on locomotor behavior.

# THE LAMPREY AS A VERTEBRATE MODEL OF SENSORIMOTOR INTEGRATION

There is large interest in understanding the neural basis of behavior. The use of lampreys as a model system has made it possible to bridge the gap between cellular mechanisms and behavior. Indeed, the lamprey nervous system is remarkably similar to the mammalian nervous system, but it contains considerably fewer neurons and is thus greatly simpler. Moreover, lamprey brainstem command cells are more easily accessible for electrophysiological studies, which can be readily combined with imaging techniques in controlled in vitro conditions with the entire locomotor circuitry intact. The supraspinal mechanisms responsible for initiating and controlling locomotion can be studied with an array of in vitro techniques, with the added benefit of including all relevant structures needed for locomotor control, and the ability to monitor ongoing swimming behavior in a semi-intact preparation consisting of the exposed brain and rostral SC with the rest of the body left intact. As such, the lamprey model has paved the way for important discoveries, including the first detailed characterization of a vertebrate CPG for locomotion (Buchanan and Grillner, 1987). Acquisition of detailed knowledge of its motor circuitry opened the way for rapid progress in understanding sensorimotor integration at the system and cellular levels. For instance, new information on sensory-evoked locomotion was provided by describing the cellular mechanisms underlying the transformation of cutaneous inputs into locomotor output at the supraspinal level (Viana Di Prisco et al., 1997, 2000; Antri et al., 2009). For the first time in any vertebrate species, the neural substrate responsible for the transformation of olfactory inputs into a locomotor output was identified using the lamprey model (Derjean et al., 2010). Overall, the lamprey nervous system is ideally suited for the mechanistic study of sensorimotor integration.

# THE NEURAL CONTROL OF LOCOMOTION

As indicated above, the basic muscle synergies responsible for locomotor propulsion are generated by SC networks collectively known as CPGs (reviewed in Grillner et al., 2008). CPGs are also involved in generating respiration (reviewed in Del Negro et al., 2002) and mastication (reviewed in Westberg and Kolta, 2011). The neuronal activity is produced by integrating the intrinsic properties of the CPG neurons and the synaptic connectivity of the inextricably linked neural network (Marder and Thirumalai, 2002; Alford et al., 2003). Synaptic activity, whether mediating the release of fast acting neurotransmitters such as glutamate or neuromodulators such as the monoamines, dopamine or 5-HT, modifies the intrinsic properties. The neural network of the lamprey locomotor CPG has been well characterized (Buchanan and Grillner, 1987). Ipsilateral glutamatergic excitation in conjunction with contralateral inhibition play a crucial role (Grillner and Wallén, 1980; Buchanan and Cohen, 1982; Brodin et al., 1985; Alford and Williams, 1989; Hellgren et al., 1992); They generate ventral root (VR) bursting that alternates across the SC (Grillner et al., 1995). The spinal locomotor circuit is activated by descending commands and in particular by glutamate release from brainstem RS cells (Buchanan et al., 1987; Ohta and Grillner, 1989). The intensity of input from RS axons regulates the frequency of these bursts of activity and therefore the speed of locomotion (Viana Di Prisco et al., 2000; Brocard and Dubuc, 2003), which may range from 0.1 to 10 Hz. Experimentally, locomotor CPG activity in the SC may be activated by stimulating the lamprey brainstem in semi-intact preparations which generate RS output (McClellan and Grillner, 1984; Sirota et al., 2000; Brocard and Dubuc, 2003; Le Ray et al., 2003) or alternatively by applying glutamate receptor agonists in isolated SCs (Cohen and Wallén, 1980; Grillner et al., 1981). The alternating pattern of VR bursting recorded under these experimental conditions is referred to as ''fictive locomotion'', and drives the coordinated contraction of muscles necessary for lamprey swimming.

The principal neurotransmitter that activates spinal CPGs is glutamate. Work in lampreys (Grillner et al., 1981; Buchanan et al., 1987; Marder, 1994), Xenopus tadpoles (Dale and Roberts, 1984; Roberts and Alford, 1986; Marder and Thirumalai, 2002; Alford et al., 2003), newborn rats (Armstrong, 1986; Kudo and Yamada, 1987) and cats (Shik et al., 1966; Douglas et al., 1993; Sirota et al., 2000) demonstrates that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor mediated transmission in the SC activates and maintains locomotion. These data are supported by direct recordings of EPSPs onto motoneurons and premotoneurons (Dale and Roberts, 1985; Brodin et al., 1988; Noga et al., 2003; Dubuc et al., 2008) and pharmacological manipulation of the resultant behaviors (Dale and Roberts, 1984; Brodin and Grillner, 1985; Cazalets et al., 1992; Chau et al., 2002; Rybak et al., 2006). Glutamatergic neurotransmission in the SC both directly excites neurons of the CPG, but may also activate either plateau properties of spinal cells as shown in the turtle (Hounsgaard and Kiehn, 1985; Guertin and Hounsgaard, 1998), or complex oscillatory properties in these neurons mediated by NMDA receptor voltage dependency and Ca2<sup>+</sup> permeability. NMDA receptor-evoked neuronal oscillations were first shown in lamprey (Sigvardt et al., 1985; Wallén and Grillner, 1987), and were since identified in mammals (Hochman et al., 1994a,b; Wilson et al., 2005; Masino et al., 2012; for a review, see Schmidt et al., 1998).

The identity of the descending glutamatergic RS command neurons is well-defined in lampreys (**Figure 1A**; Dubuc et al., 2008). RS cells have been described anatomically and physiologically. They constitute about 90% of the neurones projecting to the SC (Swain et al., 1993; Bussières, 1994; Davis and McClellan, 1994a,b). RS cells are located in one mesencephalic reticular nucleus (MRN) and three rhombencephalic reticular nuclei, the anterior (ARRN), the middle (MRRN) and the posterior (PRRN; Nieuwenhuys, 1972, 1977; Brodin et al., 1988; Swain et al., 1993; Davis and McClellan, 1994a,b). There are about 1250 RS cells on each side and about 85% of these are located in the PRRN and MRRN (Bussières, 1994). Numerous attempts to establish homologies of these nuclei to reticular nuclei in other vertebrate species have been made in the past (Kimmel et al., 1982; Cruce and Newman, 1984; ten Donkelaar et al., 1987; Nieuwenhuys and Nicholson, 1998; Brocard and Dubuc, 2003; Butler and Hodos, 2005; Nieuwenhuys, 2011). Based on the cytoarchitecture, anatomical position and connections of the reticular nuclei, the following homologies were proposed: (i) the ARRN and MRRN, which contain large medially-projecting RS cells (Müller cells; Rovainen, 1967), are homologous to the superior and middle reticular nuclei of fish, amphibians and reptiles. These nuclei would be respectively homologous to the nuclei pontis oralis and caudalis of mammals; (ii) the PRRN, which contains laterally-projecting RS cells, is homologous to the inferior reticular nuclei of fish, amphibians and reptiles. This nucleus is comparable to the nuclei reticularis gigantocellularis, ventralis and magnocellularis of mammals.

It has been shown in lampreys that the axons of large RS cells make synaptic contacts with several classes of spinal neurons and some are involved in generating locomotion (Rovainen, 1974; Buchanan, 1982; Ohta and Grillner, 1989; for a review of RS pathways in mammals, see Perreault and Glover, 2013). Similarly, studies conducted in mice and zebrafish showed that genetically identified glutamatergic RS cells of the hindbrain are involved in controlling locomotion (Hägglund et al., 2010; Bretzner and Brownstone, 2013; Kimura et al., 2013). Bouvier et al. (2015) recently identified a new population of RS cells involved in stopping locomotion. These studies opened the way to the genetic dissection of RS pathways, thus leading to further examination of their evolutionary conservation. As will be outlined below, locomotor activity is produced by spinal CPGs that are activated and maintained by descending commands. During goal-directed locomotion, this activity originates in forebrain structures, including the basal ganglia (Armstrong, 1986), to activate a largely serial process in which these higher centers recruit locomotor centers including the MLR (Shik et al., 1966; Eidelberg et al., 1981; Skinner and Garcia-Rill, 1984; Bernau et al., 1991; Marlinsky and Voitenko, 1991; Sirota et al., 2000; Cabelguen et al., 2003; Musienko et al., 2008; Ryczko et al., 2016; for reviews, see Le Ray et al., 2011; Ryczko and Dubuc, 2013) and then the RS system (Orlovskiˇi, 1970a,b, 1972; Shefchyk et al., 1984; Steeves and Jordan, 1984; Garcia-Rill et al., 1986; Jordan, 1986; Garcia-Rill and Skinner, 1987a,b). The MLR exerts an excitatory influence on RS cells either directly via glutamatergic and cholinergic (nicotinic) projections to the RS cells (Brocard and Dubuc, 2003; Le Ray et al., 2003; Noga et al., 2003; Grillner et al., 2008; Brocard et al., 2010) or indirectly via a cholinergic projection to glutamatergic muscarinoceptive cells of the brainstem that project back to

FIGURE 1 | Schematic representation of the brain and sensory-locomotor circuitry in lampreys. (A) The lamprey central nervous system (CNS). AOO, Accessory olfactory organ; Di, Diencephalon; DR, Dorsal root; Hb, Habenula; LPal, Lateral pallium; MOE, Main olfactory epithelium; OB, Olfactory bulb; OT, Optic tectum; pc, Posterior commissure; Pi, Pineal gland; Rh, Rhombencephalon; RS, Reticulospinal cells; SC, Spinal cord; V, Motor nucleus of the trigeminal nerve; VR, Ventral root. (B) The somato-locomotor pathway (purple) involves only a single relay, located in the alar plate, between the afferent sensory fibers and the RS cells. The inputs from the head region are relayed to RS cells by neurons located in the nucleus of the descending root of the trigeminal nerve (ndV), whereas inputs from the body are relayed to RS cells by neurons located in the dorsal column nucleus (DCN) or in the octavolateralis area (OLA). (C) The olfacto-locomotor pathway (orange) consists of a projection from the medial part of the OB to the mesencephalic locomotor region (MLR) via the posterior tuberculum (PT). The MLR controls locomotion in all vertebrate species through a direct projection to the command cells for locomotion, the RS cells (beige). The RS cells, in turn, project to the spinal central pattern generators (CPGs) that generate muscle synergies responsible for locomotion.

the RS cells (Smetana et al., 2007, 2010). The involvement of the cholinergic system in the control of locomotion is still subject to debate. Indeed, MLR cholinergic inputs have an excitatory influence on RS cells, but are not essential to induce locomotion (Brocard and Dubuc, 2003; Le Ray et al., 2003; Smetana et al., 2007, 2010; Jordan et al., 2014; Roseberry et al., 2016). As such, it seems that cholinergic inputs cooperate with glutamatergic inputs to amplify and sustain the locomotor output. Further investigations of the respective roles of the glutamatergic and cholinergic systems in the control of locomotion are needed.

# SENSORIMOTOR TRANSFORMATIONS OF CUTANEOUS INPUTS

Cutaneous inputs can induce, modulate and stop locomotion in vertebrates (Duysens, 1977; Viala et al., 1978; Clarke and Roberts, 1984; McClellan and Grillner, 1984; Boothby and Roberts, 1992; Frigon et al., 2012; reviewed in Grillner, 1985; Rossignol et al., 2006). The anatomical pathways and cellular mechanisms underlying these sensorimotor transformations were first characterized in the lamprey (Dubuc et al., 1993a,b; Viana Di Prisco et al., 1997, 2000, 2005; Antri et al., 2009; Le Ray et al., 2010) and more recently in Xenopus (Buhl et al., 2012).

# The Neural Pathway: From Cutaneous Receptors to Brain Locomotor Centers

As in other vertebrates, different types of mechanoreceptors are located on the skin of lampreys (Lethbridge and Potter, 1982). These include Merkel cells (Fahrenholz, 1936; Whitear and Lane, 1981), free nerve endings, and neuromasts (Lethbridge and Potter, 1982). Neuromasts are mechanosensory organs associated with the lateral line system and will not be discussed further here (see Gelman et al., 2007). The Merkel cells are present all over the body and are particularly abundant in the epidermis of the mouth, gills and fins (Fahrenholz, 1936; Whitear and Lane, 1981; Lethbridge and Potter, 1982). As seen in other vertebrates, these microvillar cells are connected to the surrounding epidermal cells by desmosomes and granules are concentrated at the site of apparent synaptic junctions with afferent nerve fibers. However, in the lamprey, their association with a nerve fiber is unique by the presence of a spur on the neurite (Whitear and Lane, 1981). Nerve fibers conveying cutaneous input enter the CNS by the dorsal roots (DRs) of the SC (body inputs) and by the trigeminal nerve (head inputs) (**Figure 1B**; Martin and Wickelgren, 1971; Matthews and Wickelgren, 1978; Rovainen and Yan, 1985; Christenson et al., 1988). Afferent fibers of the DRs and trigeminal nerve have their cell bodies within the brain/SC (dorsal cells) or in the DR ganglia/trigeminal ganglia (Rovainen and Yan, 1985). Most DR afferent fibers carrying somatosensory information from the body ascend, via the dorsal column, to terminate in the dorsal column nucleus (DCN) in the brainstem (**Figure 1B**; Dubuc et al., 1993b). Some fibers, however, continue further rostrally and reach the octavolateralis area (OLA; **Figure 1B**; Ronan and Northcutt, 1990; Dubuc et al., 1993b). Alar plate neurons from the DCN and OLA then project to the RS cells thus providing a disynaptic pathway for cutaneous inputs to reach the RS cells (**Figure 1B**; Dubuc et al., 1993b; Pflieger and Dubuc, 2004). Similarly, there is a disynaptic pathway relaying head somatosensory information to the RS cells (**Figure 1B**). After entering the brain by the sensory root of the trigeminal nerve, trigeminal afferent fibers form the descending root of the trigeminal nerve (dV) that extends down to the rostral SC. There is no well-defined sensory nucleus of the descending root (ndV) in lampreys but neurons scattered among the dV fibers constitute a diffuse ndV (Northcutt, 1979; Viana Di Prisco et al., 2005). Interestingly, tract-tracing experiments showed that some of these neurons project to the RS cells and thus could constitute a trigeminal sensory relay to RS cells (Viana Di Prisco et al., 2005).

# The Neural Mechanisms

Cutaneous primary sensory neurons (ganglion and dorsal cells) have been classified as touch, pressure, and possibly nociception, based on their response patterns to the skin stimulation. Touch cells are fast-adapting cells that respond to light mechanical stimulation of the skin with one or two spikes at the onset and offset of the stimulation (Martin and Wickelgren, 1971; Matthews and Wickelgren, 1978; Christenson et al., 1988). Pressure cells respond to mechanical stimulation of the skin by a slowly-adapting discharge with frequency related to the stimulus intensity. A third type of cell, the nociceptive cell, has been reported to respond to heavy pressure applied on the skin by a slowly-adapting discharge (Martin and Wickelgren, 1971; Matthews and Wickelgren, 1978; Rovainen and Yan, 1985). Activation of these primary sensory neurons by mechanical stimulation of the skin or electrical stimulation of the nerves (trigeminal or spinal DRs) induces post-synaptic potentials in intracellularly recorded RS cells (Viana Di Prisco et al., 1997, 2000, 2005). This finding is in accordance with behavioral observations showing that skin stimulation elicits escape swimming in intact animals (McClellan, 1988; Cardin et al., 1999). Excitatory and inhibitory amino acids are involved in the transmission of cutaneous inputs to RS cells (Dubuc et al., 1993a,b; Viana Di Prisco et al., 1995, 2005). The transmission from cutaneous (trigeminal and dorsal column) sensory afferent fibers to alar plate relay neurons was shown to be glutamatergic (Dubuc et al., 1993a,b; Viana Di Prisco et al., 1995, 2005). Cutaneous inputs are then relayed to RS cells by glycinergic and glutamatergic neurons of the relay nuclei (OLA and DCN for body inputs; dV for head inputs).

Further examination of the physiology of this disynaptic pathway led to a very interesting discovery on how RS cells transform a brief sensory input into a long-lasting motor output due to intrinsic plateau properties of RS cells (Viana Di Prisco et al., 1997). Indeed, the skin stimulation intensity—RS cell response intensity relationship is not strictly linear. At low intensities, skin stimulation elicits graded postsynaptic potentials in RS cells in a linear fashion. As the sensory stimulus intensity reaches a high level, the excitatory response in RS cells switches from sub-threshold to a large sustained depolarization that triggers escape locomotion in a semi-intact preparation. The sustaining depolarization is NMDA receptor-dependent and Ca2<sup>+</sup> entry into the cell in turn activates a Ca2+-activated non-selective cation current (ICAN). The sustained depolarizations often last for a very long duration (up to minutes). It was found that synaptic inputs could feed back onto intrinsic properties to temporally amplify the sustained depolarizations (Antri et al., 2009). Reversibly blocking SC inputs to RS cells markedly reduced the duration of the sustained depolarizations. In addition, pressure ejecting ionotropic glutamate receptor blockers on a recorded RS cell during the sustained depolarization reduced both their amplitude and duration. These findings indicate that excitatory synaptic inputs cooperate with intrinsic properties to prolong the sustained depolarizations (Antri et al., 2009). Whether similar mechanisms are involved in transforming sensory inputs from other sources into motor output remains to be determined.

# SENSORIMOTOR TRANSFORMATIONS OF OLFACTORY INPUTS

Olfactory cues can induce locomotion in vertebrates (Hasler and Wisby, 1951; Fady et al., 1998; Varendi and Porter, 2001; Johnson et al., 2009; for a review, see Daghfous et al., 2012). However, the neural substrate underlying olfactory-activated locomotion has long eluded characterization. Pioneering work (Grimm, 1960; Døving and Selset, 1980) showed that electrical stimulation of the olfactory tracts in fishes induced stereotyped motor behaviors including locomotion. Moreover, recent investigations of neural circuits and cellular mechanisms in the sea lamprey have unraveled how olfactory inputs can initiate locomotion (Derjean et al., 2010).

# The Neural Pathway: From Olfactory Sensory Neurons to Brain Locomotor Centers

In lampreys, a single nostril located along the midline on the dorsal surface of the head, anterior to the eyes opens into a single nasal cavity containing the peripheral olfactory organ. The walls of this cavity form folds or lamellae lined by an epithelium containing olfactory sensory neurons (OSNs). These lamellae house the main olfactory epithelium (MOE), and contain three ciliated OSN morphotypes: tall, intermediate and short OSNs (Laframboise et al., 2007). Their shapes and locations are similar to OSN morphotypes present in teleost fishes (Hansen and Zielinski, 2005). Diverticula (i.e., epithelial vesicles) of the MOE, mainly located in the caudoventral part of the olfactory organ, form the accessory olfactory organ (AOO) of lampreys (Scott, 1887; Leach, 1951; Hagelin and Johnels, 1955). The lumina of the AOO diverticula are linked to the lumen of the olfactory organ by tiny ducts (Hagelin and Johnels, 1955), and the cuboidal epithelium lining the AOO vesicles contains ciliated short OSNs with a broader surface than the short OSNs in the MOE (Ren et al., 2009; Chang et al., 2013). Axons extend from both MOE and AOO into the underlying lamina propria, where small axonal bundles gather to form the olfactory nerve. These OSN axons enter the OB, the primary olfactory center of the brain, where synaptic contacts are made onto the second order olfactory neurons, the OB projection neurons (the equivalent of the ''mitral/tufted'' cells of mammals). Axons from AOO OSNs terminate only in the medial part of the OB whereas axons from MOE OSNs terminate in non-medial parts of the OB and possibly sparsely in the medial part of the OB (Ren et al., 2009). Interestingly, OSN axons extending into medial and non-medial regions of the OB have distinct biochemical properties (Frontini et al., 2003). Moreover, projection neurons located in the medial and non-medial part of OB have nonoverlapping receptive fields and exhibit differences in size and dendritic morphology (Green et al., 2013). The projection neurons send their projections to third order olfactory neurons in different parts of the brain. Structures receiving these secondary olfactory projections are located mainly in the telencephalon, but some secondary olfactory fibers extend to the mesodiencephalic boundary. Third order olfactory neurons are located in the septum, striatum, pallium, habenula, hypothalamus as well as the posterior tuberculum (PT), a ventrocaudal region of the diencephalon. Tract-tracing experiments revealed that the olfactory connection to the PT originates exclusively from the medial projection neuron population (**Figure 1C**), whereas connectivity to the other aforementioned areas arise from nonmedial projection neurons. Conversely, the PT appears to be the only target of the projection neurons of the medial OB (**Figure 1C**; Figure 5 in Derjean et al., 2010; Green et al., 2013). This OB projection to the PT is of special interest as it was previously shown that the PT sends downward inputs to the MLR (**Figure 1C**; Ménard et al., 2007). The MLR is a crucial motor center located at the border between the mesencephalon and the pons. In all vertebrate species, it controls locomotion in a graded fashion, via projections to RS cells (for reviews, see Dubuc et al., 2008; Ryczko and Dubuc, 2013). Thus, the projection from the medial OB to the PT provides a way for olfactory inputs to influence locomotion in a very direct fashion (**Figure 1C**).

# The Neural Mechanisms

The activation of OSNs by chemical stimuli constitutes the first step of any olfactory-mediated behavior. In lampreys, OSNs have been shown to respond to three major classes of chemical stimuli: amino acids, steroids, and bile salts (Li et al., 1995). Stimulation of the olfactory epithelium with some of these naturally occurring olfactory stimuli can induce sustained depolarizations with spiking activity in RS cells. The stimulatory molecules include the sex pheromones 3-keto petromyzonol sulfate and 3-keto allocholic acid as well as odors such as taurocholic acid and L-arginine (Figure 1 in Derjean et al., 2010). Similarly, electrical stimulation of the olfactory nerve elicits excitatory synaptic responses in intracellularly recorded RS cells (Wickelgren, 1977; Brodin et al., 1988; Figure 2 in Derjean et al., 2010). Responses occur on both sides with a latency of around 100 ms. Calcium imaging experiments confirmed this finding by showing that repetitive stimulation of the olfactory nerve increases intracellular calcium in many RS cells (Figure 2 in Derjean et al., 2010), a sign of long-lasting afterdischarges in these cells (Viana Di Prisco et al., 1997). Local injections of glutamate antagonists in the OB blocked the responses of RS cells to olfactory nerve stimulation, indicating that synaptic transmission between OSNs and projection neurons relies on glutamate (Figure 2 in Derjean et al., 2010). The glutamatergic nature of this synapse was confirmed by showing that glutamate injection into the OB induces fictive locomotion (Figure 3 in Derjean et al., 2010). Examination of RS cell responses following the stimulation of different OB regions revealed that stimulating the medial region of the OB was more effective than stimulating non-medial regions (Figure 4 in Derjean et al., 2010). In summary, anatomical evidence and physiological experiments emphasise the role of the medial OB region in the fast relay of olfactory inputs to locomotor centers.

Because projection neurons from the medial OB region only project to the PT, the effect of PT stimulation on RS cell activity was investigated. Electrical stimulation of the PT elicited excitatory responses in RS cells with a latency of around 15 ms. Pharmacological stimulation of the PT with glutamate induced locomotor bouts in semi-intact preparations (Figure 6 in Derjean et al., 2010; Ryczko et al., 2013). Moreover, glutamate antagonist injections into the PT abolished RS cells responses to olfactory nerve stimulation, demonstrating that the OB projections to the PT are glutamatergic and that the PT is involved in olfactomotor transformations (Figure 7 in Derjean et al., 2010). Because the MLR receives inputs from the PT (Ménard et al., 2007) and projects to RS cells (Sirota et al., 2000), it is an ideal candidate to relay PT olfactory inputs to RS cells. Physiological data support this hypothesis. Local injections of glutamate antagonists into the MLR block RS cells responses to olfactory nerve stimulation (Figure 7 in Derjean et al., 2010). In addition to driving the MLR via a glutamatergic projection, the PT also modulates its activity through a dopaminergic projection. It was shown recently that stimulation of the PT induces a dopamine release in the MLR, which increases the locomotor output by a D1 receptormediated mechanism (Ryczko et al., 2013). This dopaminergic drive seems to build on the glutamatergic drive to amplify the overall PT input onto the MLR. It remains to be shown, however, whether PT dopaminergic neurons are actually recruited by olfactory inputs from medial OB projection neurons. In turn, the MLR activates RS cells (Brocard and Dubuc, 2003; Le Ray et al., 2003; Brocard et al., 2010). As such, the MLR plays a key central role in initiating and controlling locomotion (Sirota et al., 2000; Le Ray et al., 2011; Ryczko and Dubuc, 2013) induced by olfactory inputs and finely tunes the power of the locomotor output.

# COMPARISON OF THE TWO SENSORIMOTOR SYSTEMS

The activation of locomotion by the two sensory modalities, cutaneous mechanoreception and olfaction, relies on the activation of RS cells in the brainstem. Cutaneous inputs activate RS cells through relay cells located in the lateral part of the hindbrain or in the dorsal column nuclei. Olfactory inputs activate RS cells through the MLR. It is well documented that the MLR activates RS cells in a graded fashion and this results in a graded locomotor output. On the other hand, cutaneous inputs generate sustained depolarizations in RS cells in an all-or-none fashion. The sustained depolarizations were recently shown to rely on intrinsic properties (ICAN) as well as glutamatergic synaptic transmission (Viana Di Prisco et al., 2000). Ca2<sup>+</sup> imaging experiments indicate that the large RS cells are activated by both olfactory and cutaneous inputs (Viana Di Prisco et al., 1997, 2000; Derjean et al., 2010), indicating that these RS cells play a crucial role in activating locomotion induced by either sensory modality. The mechanism by which the same RS cell population could elicit graded vs. all-or-none locomotor responses have not been elucidated. It is tempting to propose that intrinsic plateau properties could be inhibited by the MLR, which is known to excite RS cells via two neurotransmitters, glutamate and acetylcholine (Le Ray et al., 2003). For instance, cholinergic inputs could suppress NMDA-induced sustained depolarizations in RS cells. There are many other possible mechanisms. The level of excitation from the MLR could be insufficient to activate the intrinsic plateau properties in RS cells, as shown in the cat preparation for sensory inputs (Brownstone et al., 1994). Another possibility would be that the intrinsic plateau properties are activated by an unidentified synaptic input originating from the cutaneous sensory inputs and not the MLR. Further work is needed to decipher between these different options.

# 5-HT MODULATION

The general organization of the serotoninergic system in lampreys is relatively well described (Pierre et al., 1992; Antri et al., 2006; Barreiro-Iglesias et al., 2009) and it is similar to that of mammals. 5-HT modulates sensory transmission at different levels in the nervous system (**Figure 2**, left). For instance, several studies have shown that 5-HT modulates sensory transmission in the SC. In mammals, sensory transmission to superficial and deep dorsal horn neurons is either depressed by 5-HT (cat: Headley et al., 1978; Anwyl, 1990; and rat: Lopez-Garcia and King, 1996; Lopez-Garcia, 1998; Garraway and Hochman, 2001) or, in a small proportion of cases, potentiated by 5-HT (rat: El-Yassir et al., 1988; Lopez-Garcia and King, 1996). In tadpoles (Sillar and Simmers, 1994) and lampreys, 5-HT decreases the amplitude of EPSPs recorded in large secondary sensory neurons (giant interneurons) in response to stimulation of primary afferents (El Manira et al., 1997). In frog motoneurons, 5-HT also depresses the EPSPs induced by DR stimulation (Ovsepian and Vesselkin, 2006). Less is known about 5-HT effects on sensory transmission at the supraspinal level. A study in guinea pigs suggested that 5-HT depresses glutamate release from trigeminal primary afferents through presynaptic inhibition (Travagli and Williams, 1996). In the lamprey, 5-HT modulation occurs at several locations along the sensorimotor pathways.

# Serotoninergic Modulation of Cutaneous Inputs

Modulatory effects of 5-HT were also investigated on the transmission of sensory inputs in the brainstem of lampreys

(1) and cutaneous (2) inputs to lamprey RS cells seem to be modulated by 5-HT (red arrows). 5-HT also acts at the spinal level by modulating the transmission from descending RS axons on spinal neurons (3).

(**Figure 2-2**). The lamprey brainstem contains rich 5-HT innervation (Steinbusch et al., 1981). There are 5-HT fibers surrounding the cell bodies of some of the large RS cells (Viana Di Prisco et al., 1994). Moreover, there is abundant 5-HT innervation within the trigeminal descending tract (Pierre et al., 1992), where the trigeminal sensory relay cells are located (Viana Di Prisco et al., 2005). 5-HT modulation of trigeminal inputs to RS cells was investigated in the brainstem of lampreys (Antri et al., 2008). Bath application of 5-HT reduced disynaptic excitatory responses in RS cells elicited by trigeminal nerve stimulation. Similar effects were seen by local ejection of 5-HT either onto the RS cells or onto the relay cells in the lateral part of the brainstem (**Figure 2-2**). 5-HT also reduced the monosynaptic EPSPs elicited from stimulation of the relay cells that receive trigeminal inputs and project onto RS cells. 5-HT increased the threshold for eliciting sustained depolarizations in response to trigeminal nerve stimulation but did not prevent them. The 5-HT innervation on RS cells appeared to originate from 5-HT neurons in the isthmic region, and also from neurons located in the pretectum and caudal rhombencephalon (**Figure 2-2**). These results indicate that 5-HT strongly modulates sensory

Daghfous et al. Neural Control of Locomotion

transmission to neural networks involved in the control of movements.

# Serotoninergic Modulation of Olfactory Inputs

In the lamprey olfactory system, 5-HT fibers were particularly prominent in the lamina propria of the olfactory epithelium, as well as in the olfactory nerve and bulb (**Figure 2-1**; Zielinski et al., 2000; Frontini et al., 2003; Abalo et al., 2007). On the other hand, 5-HT cell bodies were restricted to the lamina propria underlying the olfactory epithelium (Figures 1–3 in Zielinski et al., 2000). The 5-HT fibers were prominent within the olfactory nerve, parallel to the axons of the OSNs, from the lamina propria to the OB. However, some 5-HT fibers were also seen adjacent to the olfactory nerve. Cross sections of the olfactory nerve revealed that the 5-HT fibers where distributed evenly among the primary olfactory afferent fibers forming the nerve. Analysis of the pathway of individual 5-HT fibers using confocal z-series showed that these fibers terminated either at the junction of the olfactory nerve and OB or in the outer OB layers (i.e., olfactory nerve layer and glomerular layer). Olfactory nerve lesions experiments showed that these 5-HT fibers originate from cell bodies located in the mucosa of the olfactory organ (Zielinski et al., 2000). On the other hand, the abundant 5-HT innervation observed in the OB inner layers (i.e., granular) was not altered after cutting the olfactory nerve, demonstrating that this innervation has a different, probably central, origin. In lampreys, 5-HT neurons are present from the diencephalon to the caudal rhombencephalon (Antri et al., 2006). The telencephalon is devoid of serotonergic cell bodies (Pierre et al., 1992). Most afferents to the OB come from the telencephalon. However, some neurons projecting to the OB are located in the diencephalon (preoptic area) and midbrain tegmentum (Northcutt and Puzdrowski, 1988). Both these regions contain 5-HT neurons (Pierre et al., 1992; Antri et al., 2006), making them prime candidates as the central source of the OB 5-HT innervation. Interestingly, the meso-rhombencephalic group of 5-HT neurons seems to be homologous to the superior raphe of mammals (Antri et al., 2006), which is known to project to the OB and gate the olfactory information flow (Petzold et al., 2009). The function of the 5-HT innervation of the lamprey olfactory system is not fully understood (Zielinski et al., 2000). However, based on ongoing work in our group (Boyes et al., 2014), on the location of the 5-HT fibers in the OB (Zielinski et al., 2000), on how 5-HT acts on other sensory systems (Antri et al., 2008), and on the role of 5-HT in olfactory processing in other vertebrates (Petzold et al., 2009), it probably acts on olfactory processes by gating the sensory inflow.

# Serotoninergic Modulation of the Spinal Motor System

Several endogenous neurotransmitters have been shown to alter the output of locomotor CPGs and to modulate cellular and synaptic properties of the neurons involved (see for instance: Barbeau and Rossignol, 1991; Schotland et al., 1996; MacLean et al., 1998; Parker and Grillner, 2000; Schmidt and Jordan, 2000; MacLean and Schmidt, 2001; Grillner and Wallén, 2002; Alford et al., 2003; Perrier et al., 2003; Svensson et al., 2003). Little is known about the mechanisms involved. However, the subject is broad and we will not review the different neurotransmitter systems in different vertebrate species.

In lampreys, 5-HT modulates the descending motor commands in the lamprey SC in addition to gating sensory inputs to RS cells. Paracrine release of 5-HT activates at least two distinct receptor subtypes at three distinct subcellular locations with transduction mechanisms converging on a single behavioral modification. It has been observed a while ago that the frequency of fictive locomotion is modulated by endogenous release of neurotransmitters within the SC (Harris-Warrick and Cohen, 1985; Christenson et al., 1989; Schotland et al., 1996; Parker and Grillner, 2000; Svensson et al., 2003). Of these modulatory neurotransmitters, 5-HT reduces the frequency of VR bursting during fictive locomotion (Harris-Warrick and Cohen, 1985). This modulation occurs if 5-HT is applied exogenously, but it is also clear that activitydependent release of 5-HT from within the SC occurs and that this release similarly reduces the frequency of the CPG output (Christenson et al., 1989). This behavioral outcome of 5-HT is due, in part, to 5-HT-mediated inhibition of a postsynaptic Ca2+-dependant K+-current (KCa2) that underlies the late after-hyperpolorization of action potentials in neurons of the CPG (Wallén et al., 1989a; El Manira et al., 1994; Wikström et al., 1995; Parker and Grillner, 2000). Separately and associated directly with the synaptic activation of NMDA receptors, 5-HT mediated inhibition of a postsynaptic KCa2 is thought to play a role in prolonging fictive locomotion bursts through prolonging the plateau of NMDA tetrodotoxin (TTX) oscillations (Wallén and Grillner, 1987; Christenson et al., 1989; Wallén et al., 1989b; Schotland and Grillner, 1993; El Manira et al., 1994; Alpert and Alford, 2013; Nanou et al., 2013). Finally, presynaptic 5-HT receptor activation filters synaptic output from both descending RS command neurons (Buchanan and Grillner, 1991; Blackmer et al., 2001) and from intraspinal excitatory interneurons (Parker and Grillner, 1999; Schwartz et al., 2005). This form of presynaptic inhibition causes an augmenting synaptic response that is initially inhibited but enhanced during bursting behavior.

## Serotonin Modulation of Ca**<sup>2</sup>**<sup>+</sup> Dependent K<sup>+</sup> Conductances (KCa2) in Spinal Neurons