Anatomical and functional evidence for trace amines as unique modulators of locomotor function in the mammalian spinal cord

The trace amines (TAs), tryptamine, tyramine, and β-phenylethylamine, are synthesized from precursor amino acids via aromatic-L-amino acid decarboxylase (AADC). We explored their role in the neuromodulation of neonatal rat spinal cord motor circuits. We first showed that the spinal cord contains the substrates for TA biosynthesis (AADC) and for receptor-mediated actions via trace amine-associated receptors (TAARs) 1 and 4. We next examined the actions of the TAs on motor activity using the in vitro isolated neonatal rat spinal cord. Tyramine and tryptamine most consistently increased motor activity with prominent direct actions on motoneurons. In the presence of N-methyl-D-aspartate, all applied TAs supported expression of a locomotor-like activity (LLA) that was indistinguishable from that ordinarily observed with serotonin, suggesting that the TAs act on common central pattern generating neurons. The TAs also generated distinctive complex rhythms characterized by episodic bouts of LLA. TA actions on locomotor circuits did not require interaction with descending monoaminergic projections since evoked LLA was maintained following block of all Na+-dependent monoamine transporters or the vesicular monoamine transporter. Instead, TA (tryptamine and tyramine) actions depended on intracellular uptake via pentamidine-sensitive Na+-independent membrane transporters. Requirement for intracellular transport is consistent with the TAs having much slower LLA onset than serotonin and for activation of intracellular TAARs. To test for endogenous actions following biosynthesis, we increased intracellular amino acid levels with cycloheximide. LLA emerged and included distinctive TA-like episodic bouts. In summary, we provided anatomical and functional evidence of the TAs as an intrinsic spinal monoaminergic modulatory system capable of promoting recruitment of locomotor circuits independent of the descending monoamines. These actions support their known sympathomimetic function.

Another group of endogenous monoamines called the trace amines (TAs), include tryptamine, tyramine, and βphenylethylamine (PEA). The TAs have structural, metabolic, physiologic, and pharmacologic similarities to the classical monoamine transmitters and are synthesized from the same precursor amino acids (Saavedra, 1989). Unlike the classical monoamines, aromatic-L-amino acid decarboxylase (AADC; also called dopa decarboxylase) is the only enzyme required to produce them. Conversion from the TAs to the monoamines does not appear to occur (Berry, 2007).
The recent discovery of trace amine-associated receptors (TAARs) establishes a mechanism by which TAs can produce effects of their own (Borowsky et al., 2001;Bunzow et al., 2001). Tyramine and PEA activate TAAR1, while PEA and tryptamine activate TAAR4 (Borowsky et al., 2001). Observations using selective TAAR1 agonists and antagonists and TAAR1 knockout mice demonstrate a clear role for TA actions on CNS TAAR1 receptors. TAAR1 activity appears to depress monoamine transport and limit dopaminergic and serotonergic neuronal firing rates via interactions with presynaptic D 2 and 5-HT 1A autoreceptors, respectively (Wolinsky et al., 2007;Lindemann et al., 2008;Xie and Miller, 2008;Bradaia et al., 2009;Revel et al., 2011;Leo et al., 2014). This supports a large classical literature on the role of TAs as endogenous neuromodulators of monoaminergic excitability and neurotransmission (Boulton, 1991;Berry, 2004).
TA-induced modulatory actions in the CNS have yet to be linked with specific trace aminergic neuronal systems. Candidate neurons include 16 anatomically segregated collections of D cells (Jaeger et al., 1984). D cells contain the essential synthesis enzyme AADC but no other monoamine synthesis enzymes, and can only synthesize the TAs from their precursor amino acids (Jaeger et al., 1983(Jaeger et al., , 1984Nagatsu et al., 1988). D1 cells associate with the lumen of the spinal cord central canal (Jaeger et al., 1983). While the function of these neurons is unknown, a morphologically similar population activates locomotor circuits in larval zebrafish (Wyart et al., 2009).
As TAAR1 mRNA is found in the spinal cord (Borowsky et al., 2001), we hypothesize that the TAs and their receptors represent an intrinsic neuromodulatory system. Here, we characterize spinal expression patterns for AADC, tyramine, TAAR1 and TAAR4, then make use of an isolated neonatal rat spinal cord preparation to examine the role of applied TAs on spinal motor circuits (Kiehn, 2006). Overall we show that the TAs can activate complex rhythmic motor behaviors, including locomotor-like activity (LLA), independent of classical monoaminergic mechanisms. Some of these results have been presented in abstract form (Gieseker et al., 2004;Gozal et al., 2006Gozal et al., , 2007.

MATERIALS AND METHODS
All experimental procedures complied with the NIH guidelines for animal care and the Emory Institutional Animal Care and Use Committee. Sprague-Dawley rats aged post-natal day (P) 0-5, P14, and adult were used.

TAAR PCR
Total RNA was extracted from male and female rat whole spinal cords (P2) using QIAGEN RNeasy Mini Kit (Cat No: 74104). We performed on-column DNase digestion using the same QIAGEN kit. Total RNA was quantified by NanoDrop 2000 (Thermo Scientific). DNase-treated total RNA (1 µg) was retrotranscribed using the iScript cDNA synthesis kit according to the instructions of the manufacturer (Bio-Rad Laboratories, Hercules, CA, USA). Platinum PCR SuperMix was used for PCR reactions following the instructions of the manufacturer (Invitrogen, Cat No: 11306-016). PCR was conducted with the following cycling program: 2 min at 95 • C of initial denaturation, followed by 37 cycles of denaturation at 95 • C for 30 s, annealing at 60 • C for all TAAR primers for 30 s (except TAAR7 and TAAR9 which were at 55 • C), and extension at 72 • C for 30 s. PCR products were size-separated by electrophoresis in ethidium bromide-stained 2% agarose gels with bands visualized by ultraviolet transillumination.

In situ hybridization
Complete rat spinal cords were dissected out and the whole cords were stored in RNAlater (Qiagen, Valencia, CA) at −80 • until use. Total RNA was extracted by using Qiagen RNeasy Mini kits (Qiagen, Valencia, CA). Five micrograms of total RNA was subject to cDNA synthesis with oligo-dT15 primer and SuperScript II Reverse transcriptase (Invitrogen, Carlsbad, CA) for 1 h at 42 • C. The reverse transcriptase was inactivated, and RNA was degraded by heating at 95 • C for 5 min. Of the 20 µl of cDNA obtained from the synthesis reaction 5 µl were directly added to the PCR reaction using a PCR Mastermix kit (Eppendorf, Hamburg, Germany) containing 1 µM gene-specific primers. The primer used in this study was designed by the Invitrogen-OligoPerfect™ Designer program (Invitrogen, Carlsbad, CA). Non-radioactive single-stranded digoxigenin cRNA probes were used for in situ hybridization using methodology reported previously (Zhu et al., 2007). Briefly, single stranded, digoxigenin-labeled antisense and sense probes are transcribed in vitro using T7 and Sp6 RNA polymerase (Promega). The probe sequence for AADC is 523-927 bp (GenBank U31884), 404 bp product. The probe sequence used for TAAR1 is 400 bp long (GenBank AF380186). Hybridization was carried out at 68 • C overnight with 3 µg/ml digoxigeninlabeled antisense cRNA probe. Sense probes were used at identical concentrations and development reaction as a negative control. Sections were washed with concentrated standard saline citrate (and then incubated with anti-digoxigenin-AP Fab fragments (1:5000, Roche) in blocking buffer overnight at 4 • C. The color development reaction was carried out in the dark and neutralized with color stop buffer (10 mM Tris, pH 5, 1 mM EDTA). Slides were then dehydrated through a series of alcohol washes, coverslipped with Vectamount (Vector Labs) and images were captured on a Nikon E800 light microscope (Nikon ACT-1 software).
(0.9%NaCl, 0.1%NaNO 2 , 1 units/1 ml heparin) followed by equal volume/body weight of Lana's fixative (4% paraformaldehyde, 0.2% picric acid, 0.16 M PO 3 ); pH 6.9. In a small subset of experiments, P25 rats with and without a transection were used. In many of the experiments, Fluorogold, which does not cross the blood brain barrier, was injected intraperitoneally 24 h prior to sacrifice to retrogradely label most spinal motoneurons (Ambalavanar and Morris, 1989;Merchenthaler, 1991). The spinal cords were then isolated and post-fixed for 1 h in Lana's fixative then cryoprotected in 10% sucrose, 0.1 M PO 3 until sectioned into 10 um thick sections on a cryostat and processed for immunohistochemistry. All incubations and washes were performed in 0.1 M PO 3 -buffered saline containing 0.3% triton X-100 (PBS-T). Tissue was washed overnight in PBS-T at 4 • C followed by incubation in primary antibody for 48-72 h. Slides were then washed three times for 30 min and incubated in secondary antibody. The concentrations for the antibodies can be found in Table 1. In all experiments, omission controls were used for the primary antibodies. In addition, tyramine, TAAR1, and TAAR4 pre-absorption controls were performed with used antibody concentrations (1:100 and 1:1000) absorbed with 20 µg/100 µl of antigen (from respective antibody suppliers) for 1 h prior to incubation. As reported by the supplier (Chemicon) the tyramine antibody has >50,000-fold selectivity over its precursor amino acid (tyrosine), >40,000 selectivity over dopamine, and 800-fold selectivity over the trace amine octopamine. Images were photographed with a Nikon (Tokyo, Japan) digital camera through a Nikon E800 microscope or using an Olympus FV1000 inverted confocal microscope. Images were processed using Corel Draw (Corel, Ottawa, Ontario, Canada).

Lipophilic dye labeling
A P7 rat was anesthetized with isoflurane via inhalation for thoracic cord transection. Following dorsal laminectomy to expose lower-thoracic segments of the cord, one section of the cord between T8 and T12 was removed using surgical microdissection scissors. One week later the cord was isolated and preserved in 2% paraformaldehyde fixative, then suspended in agarose gel and labeled with the carbocyanine dye DiI. Crystals of DiI were placed at the cut surface of ventral funiculi. The dye was allowed to diffuse to identify gray matter projections sites.

Electrophysiology
Sprague-Dawley rats P0-5 were decapitated, eviscerated, and placed in a bath containing oxygenated (95% O 2 , 5% CO 2 ) artificial cerebral spinal fluid (aCSF) containing the following (in mM): 128 NaCl, 1.9 KCl, 1.2 KH 2 P0 4 , 26 NaHCO 3 , 2.4 CaCl 2 , 1.3 MgSO 4 , and 10 glucose (pH of 7.4). The spinal cord was exposed by a ventral vertebrectomy and carefully dissected out of the body cavity leaving the dorsal and ventral roots attached. The spinal cord was secured with insect pins to a chamber with Sylgard (Dow) on the bottom. Glass suction electrodes were applied to dorsal and/or ventral roots, after which the preparation was allowed to recover for at least 1 h before experimentation at room temperature. The ventral root electroneurographic activity was amplified (10,000x), band-pass filtered at 10-3000 Hz, and digitized at 5 kHz (Digidata 1321A, 16-bit; Axon Instruments). Data was captured on a computer with the pCLAMP acquisition software (v8-9, Molecular Devices; Union City, CA). Electrophysiological data analysis was performed using pCLAMP analysis software (Clampfit) or software written inhouse using MATLAB. Statistical comparisons were made using ANOVA, Student's t-test, or paired t-test. The means is reported as mean ± SD.
Motor activity was monitored using glass suction electrodes attached to ventral lumbar roots, typically bilaterally to L2 and L5. Changes in levels of motor activity were quantified by first applying a RC high-pass filter at 1 Hz to reduce drift, then calculating the root mean square of a representative ventral root signal, and comparing 100 s periods before application of the TAs and monoamines and during their period of maximal response. Changes were expressed as a percent increase over baseline. L2 ventral root activity primarily indicates activity in flexors, while L5 ventral root activity primarily indicates activity in extensors (Kiehn and Kjaerulff, 1998). LLA was defined as an alternation between right and left L2 ventral roots, with each L2 ventral root alternating with the L5 root on the same side. LLA was analyzed using the in-house MATLAB software, SpinalMOD , which calculated the frequency, peak amplitude, and phase, which was calculated using the middle of the burst (Matsushima and Grillner, 1992).

AADC and tyramine are widely expressed in the spinal cord
In situ hybridization and immunohistochemistry carried out in adult rat lumbar spinal cord showed that AADC was detected with widespread labeling throughout the spinal cord (Figure 1). AADC expression was most notable around the central canal, the ventral funiculus, and in ventral neurons, including motoneurons. There was also some nuclear labeling as seen before (Mann and Bell, 1991). AADC immunolabeling around the central canal included the previously reported D cells (Jaeger et al., 1983;Li et al., 2014). Immunohistochemical staining was undertaken as shown in Figures 1B,C. We observed clear AADC immunolabeling in neurons associated with the central canal, in axonal projections in the ventral funiculus, and in blood vessel endothelia where AADC activity is known to be high (Hardebo et al., 1979;Nagatsu et al., 1988;Li et al., 2014;Pfeil et al., 2014).
Next, immunolabeling studies on AADC and tyramine, were conducted in the neonatal spinal cord (Figure 2). This age was chosen to match the age at which the subsequent electrophysiological studies were undertaken. AADC labeling was similar to that found in the adult. Labeled spinal neurons were most notable adjacent and ventral to the central canal, the ventral funiculus, and in ventral neurons, including putative motoneurons ( Figure 2A). As observed in the adult, but more strikingly, central canal AADC + neurons projected ventrally in a stream of cells with subsequent termination of presumed axonal projections in the most medial portion of the ventral funiculus (Figure 2A, right). AADC + neurons were also found in the lateral regions near the central canal as well as in the dorsal horn (not shown), although more rarely.
To determine whether cells at the central canal could have axonal projections to the ventral funiculus, DiI crystals were applied to various ventral funicular regions in the fixed lumbar spinal cord of a P14 rat 1 week after thoracic spinalization. When dye placement contacted the midline tract, central canalassociated cells were retrogradely labeled, confirming that cells consistent with the location of D cells can project to the ventral funicular white matter tract ( Figure 2B).
Tyramine immunolabeling was characterized by enormous variability between animals, ranging from widespread to nearabsent. This variability may reflect the dynamic sensitivity of tyramine to rates of synthesis and degradation. Expression patterns included preferential ventral horn labeling, particularly associated with the central canal and in motoneurons ( Figures 3A,B). To increase detectability and further demonstrate that tyramine immunolabeling was neuronal, we preincubated spinal cords in 100 µM tyramine for 2 h. Tyramine labeling co-localized with the neuron specific marker NeuN, including in retrogradely-labeled Fluorogold + motoneurons ( Figure 3C). Tyramine commonly co-expressed with AADC in central canal D cells, in the ventral stream of cells at the midline, and in the ventral funiculus ( Figure 3D). These results are consistent with the notion that D cells and related midline neurons synthesize tyramine. We also observed co-labeling in putative spinal motoneurons ( Figure 3E).

Trace amine-associated receptor expression in the spinal cord
Given that 17 TAAR rat genes have been identified, we tested for the presence of TAAR gene expression in the spinal cord. RT-PCR was undertaken on total RNA prepared from neonatal rat spinal cord of both sexes. We observed detectable transcripts for TAARs 1-6, 7a, 8a, and 9 (Figures 4A,B). Because TAAR1 is the only receptor for which there has been detailed study, we also undertook in situ hybridization for TAAR1 in the neonate. Widespread spinal cord labeling was observed including around the central canal and in the ventral horn region associated with motoneurons ( Figure 4C).
Immunolabeling for both TAAR1 and TAAR4 was found throughout the neonatal spinal cord, including in motoneurons and in neurons around the central canal (Figures 4D,E). TAAR1 and TAAR4 labeling in all neurons appeared intracellular, consistent with previous reported results for TAAR1 (Miller, 2011). A cytoplasmic location of ligand and receptor (e.g., tyramine and TAAR1) supports intracellular activation of signal transduction pathways, as suggested previously (Miller, 2011). The presence of TAAR1 and TAAR4 provides for actions mediated by tyramine and PEA, and tryptamine and PEA, respectively (Borowsky et al., 2001;Bunzow et al., 2001).

TA ACTIONS ON MOTOR AND LOCOMOTOR ACTIVITY IN VITRO
To examine whether tyramine, tryptamine, and PEA increased motor excitability, population motor activity was recorded from a lumbar ventral root before and after their application. As shown in Figure 5A, tryptamine and tyramine increased motor activity while PEA did not. To test whether the TAs acted directly on motoneurons, chemical synaptic transmission was minimized by replacing the regular aCSF with one having high Mg 2+/ low Ca 2+ or in nominally zero Ca 2+ (Shreckengost et al., 2010). Under these conditions, dorsal root stimulationevoked reflexes were abolished (Figure 5B 1 ). Subsequently, tryptamine and tyramine still increased motor activity (Figures 5B 2 ,C).
Interestingly, tryptamine (n = 1/14), tyramine (n = 8/24), and PEA (n = 7/10) were able to generate a bursting pattern never seen with the monoamines in this preparation. The pattern was more complex with episodic bouts of bursting ( Figure 7D). During these bouts, burst frequencies were statistically indistinguishable from 5-HT locomotion. Episodic bouts of bursting were variable but usually concurrent across roots and alternated with comparatively quiescent periods at very slow average frequencies (Figure 8). For tyramine, bursting patterns interconverted between continuous locomotion to episodic bouts in one experiment. Notably, bouts of episodic bursting were the dominant activity pattern observed for PEA ( Figure 7C). Expressed bouts of episodic bursting were highly variable, containing up to 47 events within an episode and with quiescent periods varying from 5 to 230 s.
Frequently, there were progressive increases and decreases in the locomotor burst amplitude within each episode, which were noticeable in at least some bouts for all TAs tested (Figure 8). Similarly, locomotor frequency could be seen to wax and wane during these bouts, supporting a sinusoidal drive to the CPG. This is highlighted in Figure 8A with progressive increases then decreases in burst amplitude, and locomotor frequency are further quantified in Figure 8D.

Differences in receptor pharmacology
5-HT is thought to activate spinal locomotor circuits via 5-HT 2 and 5-HT 7 receptors (Madriaga et al., 2004;Liu and Jordan, 2005;Liu et al., 2009). Methysergide is a high affinity non-selective 5-HT 1 , 5-HT 2 , and 5-HT 7 receptor antagonist (Madriaga et al., 2004) and a tryptamine binding site antagonist (Martin et al., 1988). To determine if the TAs and other monoamines had a similar sensitivity to methysergide as 5-HT, methysergide was added to TA and monoamine-induced rhythms (with NMDA) at progressively increasing doses ( Table 2). Tryptamine was more sensitive than 5-HT to methysergide block. Tyramine was variably sensitive to methysergide block, while PEA, NA, and DA-evoked LLA was methysergide-insensitive even at the highest dose tested (10 µM). Based on methysergide sensitivity, it appears likely that tyramine and PEA actions are not dependent on 5-HT receptor activation. Methysergide block of tryptamine-evoked LLA may be via competitive block at 5-HT receptors and/or at the methysergide-sensitive tryptamine binding site (Martin et al., 1988).

Temporal differences in activation onset
When time to burst onset after bath application was examined, 5-HT and NA had rapid burst onset (medians of 7.5 and 2.5 s, respectively). In comparison, burst onset took dramatically longer for DA, tryptamine, tyramine, and PEA (medians of 480, 300, 300, and 540 s, respectively; all are p < 0.05) (Figure 9). This suggests that the TAs and DA are not acting predominantly on plasma membrane metabotropic receptors. required interactions with the vesicular monoamine transporter (VMAT), we compared the locomotor-inducing ability of the TAs to those of 5-HT (always in the presence of NMDA) in bath chambers pre-incubated with the VMAT inhibitor reserpine (>40 min) (10 µM; Figure 10A). In the presence of reserpine, 5-HT and tryptamine evoked LLA in all cords tested (n = 5/5). In comparison, tyramine-and PEA-evoked LLA was seen in 4.5 and 3/5 cords, respectively. As reserpine has moderate binding affinity to D 2 and D 3 dopamine receptors (Toll et al., 1998) and also interferes with DAT and NET (Yamamoto et al., 2007;Mandela et al., 2010), non-specific actions including at a PEA binding site may have accounted for the reduced incidence of LLA with PEA. Overall, the VMAT inhibitor, reserpine, does not prevent the expression of TA-evoked LLA.

TA-evoked locomotor-like activity is independent of highaffinity monoamine transporters located on descending monoaminergic terminals.
The high-affinity monoamine transporters for 5-HT (SERT), DA (DAT), and NA (NET) control the extracellular concentrations of monoamines and maintain presynaptic function (Torres et al., 2003). Since the monoamine transporters are also a substrate for transport of TAs (Xie et al., 2007), a mechanism by which the TAs could exert their action is by transporter-mediated presynaptic uptake at descending monoaminergic terminals. For example, the TAs could elevate extracellular monoamines by competing for uptake or displacing biologically active biogenic amines from their storage leading to efflux via reverse transport (Premont et al., 2001). Additionally, once transported intracellularly, they could act on presynaptic TAARs to alter basal activity (Miller, 2011).
To explore this, we compared the motor rhythm-inducing actions of the TAs or monoamines to those seen in the presence of co-applied SERT, DAT, and NET transport inhibitors (citalopram, 1 µM; bupropion, 1 µM; and clomipramine, 5 µM, respectively). Given the variability in expression of TA-evoked responses observed in earlier experiments, we undertook most subsequent studies using littermate pairs of spinal cords in the same experimental chamber (Figure 10B). 5-HT-evoked LLA was not significantly altered (n = 12; 0.16 ± 0.06 Hz without vs. 0.15 ± 0.04 Hz with inhibitors). Similarly, PEA-evoked LLA was unaffected by the transport inhibitors in 9 animals (0.17 ± 0.09 Hz vs. 0.15 ± 0.06 Hz). Tryptamine-induced motor rhythms were comparable in 7/8 animals and blocked in another (0.11 ± 0.03 Hz without vs. 0.08 ± 0.06 Hz with inhibitors). Tyramine-evoked actions were blocked in 2/11 animals. Interestingly, tyramine block occurred in both spinal cords of a single chamber, littermate-paired experiment (Figure 10Bb 2 ). In the remaining 9 neonates, LLA was not blocked (0.08 ± 0.07 Hz before vs. 0.12 ± 0.10 Hz in the presence of inhibitors). Thus, the TAs can activate motor rhythms independent of actions on high-affinity monoamine transporters in the isolated spinal cord. As no spinal neurons express NET, SERT, or DAT, direct actions on spinal circuits are strongly implicated.

Intracellular transport of tyramine and tryptamine are required for locomotor-like activity: dependence on low affinity Na +independent membrane transporters.
OCTs belong to a family of Na + and Cl − independent bidirectional solute carrier (SLC) transporters, SLC22A (Jonker and Schinkel, 2004). The family contains three subtypes (OCT1-3) and all appear widely expressed in the spinal cord (Allen_Spinal_Cord_Atlas, 2009). Tyramine was shown to be the best physiological substrate for OCT1 and OCT2 (Schomig et al., 2006). In comparison, DA, NA, and 5-HT clearance was too low for a primary role in transport while tryptamine and PEA actions were not tested. The plasma membrane monoamine transporter (PMAT) is another at Na + -independent transporter with comparable pharmacological and transport properties to the OCTs. PMAT transports biogenic amines including tyramine and tryptamine (Engel and Wang, 2005) while PEA acts as a potent cis-transport inhibitor (Ho et al., 2011). PMAT has widespread expression in neurons in brain and spinal cord (Engel and Wang, 2005;Allen_Spinal_Cord_Atlas, 2009).
Pentamidine is an OCT inhibitor with IC 50 values of 0.4 and 5.1 µM for OCT1 and OCT2, respectively (Jung et al., 2008). The effects of pentamidine were only tested on tyramine and tryptamine-evoked LLA (Figure 11A). Tyramine-induced LLA (0.19 ± 0.02 Hz) was blocked in all 8/8 cords tested (p < 0.001). Tryptamine was also tested in 5 of these animals. Pentamidine completely blocked tryptamine-induced LLA in 2/5 animals with reduced overall frequency in the other 3 animals (0.16 ± 0.02 to 0.07 ± 0.07 Hz; p < 0.05). Depressant actions were associated with dramatic reductions in the magnitude of observed activity ( Figure 11B). When 5-HT was subsequently applied, LLA reemerged in all 5 animals (0.17 ± 0.08 Hz) supporting an independence of pentamidine actions on 5-HT-evoked LLA (Figure 11B). It is noteworthy that pentamidine is also a NMDA receptor non-competitive antagonist, but did not prevent expression of 5-HT/NMDA LLA (Reynolds and Aizenman, 1992;Reynolds et al., 1993). Shown is the bout highlighted by the box on r-L2 in panel A with overlaid plots of instantaneous frequency (blue) and peak amplitude of the rectified response (green). Note the trend for amplitude and frequency to increase then decrease over the episodic bout of locomotion. Events within the shaded box emphasize the dominance of higher frequency/higher amplitude values in the middle of the episode of bursting. The upper traces in gray at each lumbar root represent the rectified/low-pass filtered equivalent of the raw waveforms below them. Scale bars are 100 s in top panels and 10 s in bottom panels.

Evidence that the TAs can be endogenously synthesized from their aromatic amino acid precursors to generate locomotorlike activity.
Intrinsic spinal cord AADC activity should enable endogenous synthesis of the TAs from their precursor aromatic amino acids (AAAs), phenylalanine (for PEA), tyrosine (for tyramine), and tryptophan (for tryptamine). We therefore tested whether bath application of AAAs lead to the expression of rhythmic motor activity. Tyrosine (n = 5), tryptophan (n = 3), or phenylalanine (n = 3) were applied at doses between 100 and 200 µM with or without NMDA. In two additional experiments, the AAAs were co-applied. In all cases, no obvious maintained motor rhythms were observed (not shown).
As in vitro experiments are undertaken in the absence of essential amino acid containing media, we explored whether the lack of observed effect was related to lack of substrate availability. In the developing neonate, available cytoplasmic AAAs may be preferentially directed toward protein synthesis. We therefore tested the effects of complete arrest of protein synthesis using cycloheximide (100 µM). Cycloheximide is known to increase intracellular levels of AAA (Beugnet et al., 2003) and inhibition is mostly reversible within 1 h (Abbas, 2013).
Eight dual experiments were conducted with pairs of spinal cords from littermates recorded simultaneously in the same bath chamber (n = 16 total sample). In the presence of NMDA and cycloheximide, motor rhythms with epochs consistent with a locomotor-like coordination were observed in all 16 spinal cords. Activity was comparatively fast at 0.31 ± 0.09 Hz but appeared to be weaker and more variable than that seen with Following induced LLA, methysergide was applied in progressively increasing doses as shown to assess differential monoamines agonist sensitivity to block.
FIGURE 9 | Dopamine and the TAs take a significantly longer time to initiate bursting than 5-HT and NA. The box-and-whisker plots show the time it takes to initiate bursting (always in the presence of NMDA). The red line is the median and individual outliers, defined as more than 1.5 times the interquartile range were observed for 5-HT (cross) and for tryptamine at 1800 s (not shown). Asterisk ( * ) indicates p < 0.05. Samples sizes are shown at top. the monoamines. Cycloheximide was applied before NMDA (n = 2), after NMDA (n = 5), or co-applied with NMDA (n = 1). Cycloheximide alone produced no observable action, but episodic LLA emerged with subsequent application of NMDA ( Figure 12A). NMDA applied alone generated some epochs of weak bursting activity in at least one of the paired cords in 3/5 experiments with subsequent addition of cycloheximide leading to episodic LLA on spinal cord pairs ( Figure 12B). In the 3 experiments with only bath applied NMDA, spontaneous bursting was also seen prior to NMDA application. Overall increases in initial cord excitability seen may have been due to residual cycloheximide from prior days. In one of these experiments, NMDA alone generated robust bursting though subsequent application of cycloheximide led to a clearly strengthened and regularized LLA ( Figure 12C). As described earlier, episodic tyramine-and PEA-evoked an episodic form of LLA never seen with the classical monoamines (in 33 and 70% of animals, respectively; Figure 7). After cycloheximide, episodic bouts of LLA appeared in 69% (11/16) of tested animals, 4 of which expressed rhythms that were distinctly PEAlike ( Figure 12D). LLA coordination across recorded ventral roots is depicted in a polar plot of relative burst timing ( Figure 12E). Thus, in the absence of protein synthesis, an increased bioavailable source of amino acids enabled the expression of episodic LLA, a distinctive feature of TA-evoked motor rhythms.

DISCUSSION
The neonatal rat spinal cord was shown to contain AADC, the essential synthesis enzyme for TA biosynthesis from their amino acid precursors, as well as their cognate G protein-coupled receptors (TAAR1 and TAAR4) that together define the essential substrates to exert biological actions. Whether and how this intrinsic spinal aminergic modulatory system is recruited is unknown, but exogenously provided TAs clearly promote motor pattern generating circuits. To exert their modulatory actions TAs show dependence on intracellular translocation via Na + -independent transporters, consistent with prominent intracellularly located TAARs. Unlike the classical monoamines, the TAs can also generate an episodic form of LLA. That episodic motor rhythms are also seen following increases in precursor amino acid availability supports the endogenous TA biosynthesis as a means to promote motor circuit activation.

AADC and tyramine expression in the spinal cord
In both adult and neonate, we regularly observed AADC labeling in cells surrounding the central canal (D cells), in a ventral stream from this region, and in blood vessels. Central canal-related labeling is consistent with previous reports in rat, mouse, and monkey (Jaeger et al., 1983;Nagatsu et al., 1988;Barraud et al., 2010;Li et al., 2014;Wienecke et al., 2014). Labeling in blood vessels has also been reported (Jaeger et al., 1983;Li et al., 2014). AADC immunolabeling was also seen in other gray matter areas and in a midline ventral funiculus white matter bundle (Wienecke et al., 2014). This white matter tract likely represents axon projections of D cells as we observed that DiI applied to this region retrogradely labeled central canal cells. In the neonate, we also observed a ventral collection of AADC + midline cells emanating from the central canal. These cells may represent a ventral "migratory stream" of active neurogenesis since Wienecke et al. (2014) observed that D cells are Neun − /doublecortin + in adult rat, and thus likely to be newly generated neurons or neural precursors. Interestingly, motoneurons appeared to be weakly AADC + though neither in situ hybridization nor immunodetection of AADC has been previously reported in rat spinal motoneurons. To support this observation, we mined our earlier microarray expression profiling study on laser capture microdissected Frontiers in Neural Circuits www.frontiersin.org November 2014 | Volume 8 | Article 134 | 12

FIGURE 10 | TA-induced LLA is not principally via uptake by high-affinity plasma membrane or vesicular monoamine transporters. (A)
The vesicular monoamine transport inhibitor, reserpine (10 µM), did not prevent TA-induced modulatory actions. Shown is evoked LLA in two rats (a,b) (B) TA-evoked actions were tested before and after combined application of the transport inhibitors, citalopram (1 µM; a SERT inhibitor), buproprion (1 µM; a DAT inhibitor), and clomipramine (5 µM; a SERT and NET inhibitor). The transport inhibitors were usually without effect as shown for tryptamine (a), but could also depress (b; in this case tyramine) or facilitate LLA (c; in this case PEA). In all panels, 5-HT and the TAs were always in the presence of NMDA. Scale bars are 10 s in all panels.
medial and lateral motor column motoneurons (Cui et al., 2006). Using the present/absent call in Affymetrix Microarray Suite software, AADC cDNA was detected as present in all samples of motoneurons. In comparison, tyrosine hydroxylase and dopamine-β-hydroxylase cDNA were reported as absent.
Tyramine immunolabeling was also seen in AADC-containing neurons, consistent with AADC-mediated synthesis from tyrosine. It is assumed that tryptamine and PEA would also be synthesized by AADC from their respective amino acid precursors, but could not identify commercially available antibodies with acceptable specificity for these TAs. While the most consistent labeling observed for tyramine and AADC was in central canal midline regions and motoneurons, we noted considerable inter-animal variability. Indeed, the TAs have been described as protean. Metabolic sensitivity to temporal shifts in substrate availability may be a defining feature of the AADC-TA-TAAR modulatory system (Burchett and Hicks, 2006).

Trace amine-associated receptors 1 and 4 in the spinal cord
Previously, in situ hybridization, RT-PCR, and LacZ reporter expression studies all observed labeled TAAR1 in the brain (Borowsky et al., 2001;Lindemann et al., 2008) and one report also examined and detected expression in spinal cord (Borowsky et al., 2001). While TAARs are thought to be G s -coupled (Borowsky et al., 2001), there is also evidence of activation of G qcoupled signal transduction pathways (Panas et al., 2012). Here, RT-PCR identified expression of several TAARs in the neonatal rat spinal cord, including TAAR1 and TAAR4. We also observed TAAR1 and TAAR4 immunolabeling in neurons near the central canal and in motoneurons. TAAR1 and TAAR4 expression observed in the spinal cord overlapped with the expression of AADC and tyramine. Since tyramine and PEA activate TAAR1 and tryptamine and PEA activate TAAR4 (Borowsky et al., 2001), the TAs have a substrate for biological actions in the spinal cord. Suggestive immunolabeling evidence of a cytoplasmic location of both ligand (tyramine) and receptors (TAAR1 and TAAR4) supports an intracellular activation of signal transduction pathways (Miller, 2011). This is fully consistent with exogenously applied TAs showing dependence on transmembrane transport and taking longer than 5-HT and NA to exert modulatory actions. In summary, the mechanisms for TA synthesis and actions are anatomically coincident, and provide a substrate by which TAs can produce effects on their own.

ELECTROPHYSIOLOGICAL ACTIONS
When applied alone, the monoamines, tryptamine, and tyramine increased motor activity, including via direct excitatory actions on motoneurons. This agrees with a previous study showing that tyramine can act directly on motoneurons (Kitazawa et al., 1985). PEA is an agonist at both TAAR1 and TAAR4 receptors (Borowsky et al., 2001;Bunzow et al., 2001); so their expression in motoneurons should also facilitate motor activity. However, no overt actions were observed. One possibility is that there is no membrane transporter for PEA in motoneurons. Another possibility is that TAARs are expressed as heterodimers (e.g., TAAR1/TAAR4) that preferentially interfere with PEA binding (Babusyte et al., 2013). Other possibilities include actions on other receptors (e.g., G i -coupled α 2 -adrenergic) that compete with TAAR1 and TAAR4 mediated actions (Pacifico et al., 2012). We observed that 5-HT, NA, and DA produced comparable LLA in the presence of NMDA, consistent with reports of sublocomotor doses of NMDA helping to stabilize and regularize the locomotor rhythm (Sqalli-Houssaini et al., 1993;Cowley and Schmidt, 1994;Kjaerulff et al., 1994;Schmidt et al., 1998). The TAs also produced LLA indistinguishable from that seen with the monoamine transmitters, strongly suggests that the TAs are acting at the level of the locomotor central pattern generator (CPG) and recruiting the same pattern-generating circuits. However, the detailed pattern of activation (which CPG neuron classes are recruited) may not be the same.
The TAs were also generated an episodic form of LLA not seen with the monoamine transmitters, and both continuous and episodic locomotor phenotypes could be observed at different times within an individual animal. Episodic LLA may represent a physiologic pattern recruited by endogenous mechanisms. For example, voluntary wheel running in rats and mice is episodic, occurring in short bouts separated by longer periods (Hanagasioglu and Borbely, 1982;Eikelboom and Mills, 1988;De Bono et al., 2006). Whelan et al. (2000) reported spontaneous episodes of rhythmic ventral root activity in the mouse very similar to those seen here (Whelan et al., 2000).
Episodic LLA patterns may reflect recruitment of additional neurons or intrinsic membrane properties that influence the output of the spinal locomotor CPG. This is explored theoretically Following pre-incubation of cycloheximide (1), applied NMDA leads to the expression of LLA in both cords (2). Replacement of solution and subsequent application of NMDA (3) reinstates some bursting in cord, but a full LLA pattern emerges in both cords only after reapplication of cycloheximide (4). (B) NMDA generated some spiking in both cords of this pair (1) while addition of cycloheximide led to LLA in both pairs. (C 1 ) In this pair, some spontaneous excitability was seen initially (1) and NMDA + cycloheximide led to coordinated LLA (2). Note the overall similarity in response in paired spinal cords. (C 2 ) Low-pass filtered recording of additional recorded LLA activity in rat 2 after cycloheximide and NMDA. L-L2 activity (black) is inverted and adjacent to r-L2 to show anti-phase left-right coordination between flexors, and overlapped with r-L5 activity to show phase coupling with contralateral extensor activity. (D) Comparison to PEA evoked rhythm. Longer duration recordings with rectified, low-pass filtered trace compare PEA expression pattern to cycloheximide/NMDA LLA in two separate animals. Scale bar is 10 s in all (A-D). (E) Phase relationship of r-L2 burst onset (from 0 • ) to l-L2 and r-L5 burst onsets (blue and red symbols, respectively). The cycle progresses clockwise from 0 • (in-phase) to 180 • (out-of-phase) to 360 • /0 • . Events correspond to bursts in lower record of (D) identified with vertical dotted bars. Timing is consistent with LLA. Arrow length represents the concentration (r) about the mean angle (Ø). The inner circle inside the phase diagram denotes the critical r vector calculated from the Rayleigh's Z table using α = 0.05 (Zar, 1999 in Figure 13A with the assumption that the TA-induced regular LLA pattern is produced at the CPG level. Episodic modulation of LLA might occur in neurons that project onto the CPG (left illustration) themselves producing a much slower rhythmic synaptic drive onto the CPG. This is consistent with observed waxing and waning of LLA. Obvious candidate neurons are the AADC-expressing D cells surrounding the central canal (described below). Other possible organizations are depicted and explored in Figure 13A.
While the function of these D cells in the mammal is still unknown, similarly-located and projecting CSF-contacting neurons in larval zebrafish initiate slow swimming by optogenetic stimulation. Their genetic silencing reduced the frequency of spontaneous locomotion, and they provided the necessary tone for spontaneous forward swimming (Wyart et al., 2009). Based on the similarity in location, it is not unreasonable to consider a comparable role for D cells in the mammal. Aside from CSFcontacting D cells, many other AADC + neurons were located in adjacent ventromedial locations. This location is consistent with ventromedially located interneurons shown to undergo intrinsic membrane voltage oscillations including in association with neurochemicals that induce LLA (Hochman et al., 1994;Tazerart et al., 2008;Brocard et al., 2013). Thus, D cells, with projections into the ventral funiculus, and previously reported synaptic projections toward motor nuclei (Jaeger et al., 1983) represent a likely source of the episodic modulation of locomotor activity.

TA ACTIONS ARE MECHANISTICALLY DISTINCT FROM THE MONOAMINES TRANSMITTERS
The TAs are structurally similar to the classical monoamines and may act as monoamine receptor agonists. Tryptamine can activate 5-HT 2 and 5-HT 7 receptors (Boess and Martin, 1994), on which 5-HT induced locomotion is dependent (Madriaga et al., 2004;Liu and Jordan, 2005;Liu et al., 2009), and tryptamine and 5-HT evoke similar locomotor patterns with similar sensitivity to methysergide block. On the other hand, tyramine and PEAevoked LLA appear to have very low affinity to the monoamine receptors (U'Prichard et al., 1977;Shen et al., 1993;Peddi et al., 2003). Critically, the TAs including tryptamine require 50-100fold longer incubations periods to activate LLA and this difference is not consistent actions via plasma membrane monoamine receptors. Therefore, tryptamine may require activation of 5HT as well as TAARs to generate LLA.
Slower actions were not due to indirect release of monoamines through their transporters since TA-induced LLA remained following block of VMAT and the Na + -dependent monoamine transporters. Instead, prolonged incubations periods are consistent with observed dependence of tyramine-and tryptamine-evoked LLA on intracellular transport via low affinity Na + -independent membrane transporters. Collectively these observations support TA recruitment of LLA by intrinsic spinal mechanisms independent of descending monoaminergic systems.
Notably, DA also took much longer to initiate LLA. Since DA can be generated in endothelia subsequent to hypoxic stress (Pfeil et al., 2014) and DA is a low affinity agonist at Putative trace aminergic neurons are the AADC + D cells. Their location is within the spinal region responsible for locomotor rhythmogenesis. Their effect could be to generate a waxing and waning of CPG output to motoneurons (MN). (2) Modulatory actions within a subclass of CPG neurons. For example, the synaptic drive provided by the CPG could interact with slow frequency intrinsic voltage oscillations. (3) Modulatory actions within motoneurons are not consistent with observed waxing and waning in locomotor CPG frequency. (B) Putative transport and intracellular signaling mechanisms for TA actions. The TAs, tyramine and tryptamine, undergo Na + -independent membrane transport via the PCTs and PMAT, respectively. PEA transport could include the LATs, which are also responsible for transport of the precursor aromatic amino acids (AAAs), tyrosine, phenylalanine, and tryptophan. It is also possible for these TAs to be made from their AAA precursors in AADC-containing cells. Once inside, the TAs act on intracellularly-located TAARs to produce Gs-coupled neuromodulatory responses. The TAs could also be generated via AADC in some neurons (here neuron A) or from endothelia then transported into adjacent non-AADC but TAAR-containing neurons to exert neuromodulatory actions. Abbreviations: PMAT, plasma membrane monoamines transporter; OCTs, organic cat ion transporters; LATs, large neutral amino acid transporters; PKA, protein kinase A; cAMKII, calcium/calmodulin dependent protein kinase II; AC, adenylate cyclase; 5-HTR, 5-HT receptor; TA, trace amine; TAAR, trace amine-associated receptor.

Frontiers in Neural Circuits
www.frontiersin.org November 2014 | Volume 8 | Article 134 | 16 TAAR1 in rat (Bunzow et al., 2001), DA-induced LLA may normally associate with vascular release and subsequent intracellular neuronal transport via PMAT. Dependence on transmembrane transport for PEA-evoked actions was not tested. Biologically, PEA transport is known to occur via a saturable proton-dependent transport process independent of all currently studied OCTs (Fischer et al., 2010). PEA is also a potent cis-transport inhibitor of PMAT (Ho et al., 2011). Some PEA transport may occur by Na + -independent system L amino acid transporter LAT3 (Babu et al., 2003), which shows widespread expression in the mouse spinal cord (Allen_Spinal_Cord_Atlas, 2009), as well as by simple diffusion (Berry et al., 2013).
As reported for TAAR1 in HEK cells (Bunzow et al., 2001;Miller, 2011), we observed cytoplasmic labeling for TAAR1 and TAAR4, both of which are activated by the TAs (Borowsky et al., 2001). A cytoplasmic location of the ligand and the receptor (e.g., tyramine and TAAR1) would support intracellular activation of signal transduction pathways (Miller, 2011). Such a co-localization would not require release from vesicles and could explain why the TAs do not appear to be found there (Berry, 2004;Burchett and Hicks, 2006).
Putative transport mechanisms for the TA are shown in Figure 13B. The TAs, tyramine and tryptamine, can transport via OCTs and PMAT, respectively, whereas PEA may transport via LAT3 but more likely via a currently unidentified transporter. TA biosynthesis can alternatively be generated intracellularly from aromatic amino acid (AAA) precursors in AADC-containing cells to act on intracellularly-located TAARs. This would constitute a form of biochemical integration (Katz and Clemens, 2001). TAs generated via AADC in some neurons (here neuron A) or from endothelia can transport into adjacent non-AADC but TAARcontaining neurons to exert neuromodulatory actions (OCTs and PMAT transport is bidirectional). Last, as AADC and TAARs are present in vasculature, TA modulation of vascular function may lead to secondary actions on neurons (Hardebo et al., 1979;Nagatsu et al., 1988;Broadley, 2010;Anwar et al., 2012).

TA BIOSYNTHESIS AS A SYMPATHETIC AUTONOMIC CELLULAR STRESS RESPONSE
Intrinsic spinal cord AADC activity should enable endogenous synthesis of the TAs from their AAA precursors. However, when the AAAs were bath-applied, no obvious maintained motor rhythms were observed. Since intracellular transport of AAAs may be rapidly sequestered for protein synthesis during this neonatal period of dramatic growth, we blocked protein synthesis with cycloheximide in order to increase biochemically access to AAAs (Beugnet et al., 2003). The effects of cycloheximide were dramatic with LLA developing in all animals. Additional studies are required to directly link increases in AAAs to increases in endogenous TA biosynthesis to induce a neuromodulatory response. However, emergent LLA included TA-like episodic bouts including those with burst structures notably comparable to that seen with PEA.
Access to precursor amino acids for TA biosynthesis may depend heavily stress-induced protein catabolism. As the TAs are known sympathomimetics (Branchek and Blackburn, 2003;Berry, 2004), spinal TAs may function to facilitate motor responses during strong sympathetic nervous system activation. For example, marked PEA increases are seen in human urine after a highly stressful event (Paulos and Tessel, 1982). Just as DA can be generated via AADC in endothelia subsequent to hypoxic stress (Pfeil et al., 2014), intracellularly-synthesized TAs may comprise an integral physiological component of the autonomic stress response.